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Biosynthesis of the modified tetrapyrroles—the pigments of life

  • Donald A. Bryant
    Correspondence
    Studies in this author's laboratory were supported by Photosynthetic Systems Program, Division of Chemical Sciences, Geosciences, and Biosciences (CSGB), Office of Basic Energy Sciences of the United States Department of Energy Grant DE-FG02-94ER20137 and United States National Science Foundation Grant MCB-1613022. To whom correspondence may be addressed.
    Affiliations
    Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802

    Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717
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  • C. Neil Hunter
    Correspondence
    Supported by Biotechnology and Biological Sciences Research Council (UK) Grant BB/M000265/1. To whom correspondence may be addressed.
    Affiliations
    Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom
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  • Martin J. Warren
    Correspondence
    Supported by Biotechnology and Biological Sciences Research Council (UK) Grants BB/N00924X/1 and BB/S002197/1. To whom correspondence may be addressed.
    Affiliations
    School of Biosciences, University of Kent, Canterbury CT2 7NJ, United Kingdom

    Quadram Institute Bioscience, Norwich Research Park, Norwich NR4 7UQ, United Kingdom
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Open AccessPublished:April 02, 2020DOI:https://doi.org/10.1074/jbc.REV120.006194
      Modified tetrapyrroles are large macrocyclic compounds, consisting of diverse conjugation and metal chelation systems and imparting an array of colors to the biological structures that contain them. Tetrapyrroles represent some of the most complex small molecules synthesized by cells and are involved in many essential processes that are fundamental to life on Earth, including photosynthesis, respiration, and catalysis. These molecules are all derived from a common template through a series of enzyme-mediated transformations that alter the oxidation state of the macrocycle and also modify its size, its side-chain composition, and the nature of the centrally chelated metal ion. The different modified tetrapyrroles include chlorophylls, hemes, siroheme, corrins (including vitamin B12), coenzyme F430, heme d1, and bilins. After nearly a century of study, almost all of the more than 90 different enzymes that synthesize this family of compounds are now known, and expression of reconstructed operons in heterologous hosts has confirmed that most pathways are complete. Aside from the highly diverse nature of the chemical reactions catalyzed, an interesting aspect of comparative biochemistry is to see how different enzymes and even entire pathways have evolved to perform alternative chemical reactions to produce the same end products in the presence and absence of oxygen. Although there is still much to learn, our current understanding of tetrapyrrole biogenesis represents a remarkable biochemical milestone that is summarized in this review.

      Introduction

      Modified tetrapyrroles play essential roles in a broad range of essential biological processes. Their large macrocyclic structures and diverse conjugation and metal chelation systems also provide an array of colors, such that they have been dubbed the “pigments of life” (
      • Battersby A.R.
      Tetrapyrroles: the pigments of life.
      ). These life pigments include the hemes, chlorophylls (Chls),
      The abbreviations used are: Chl
      chlorophyll
      BChl
      bacteriochlorophyll
      ALA
      aminolevulinic acid
      GSA
      glutamate semialdehyde
      PBG
      porphobilinogen
      HMB
      hydroxymethylbilane
      ProtoIX
      protoporphyrin IXα
      Chlide
      chlorophyllide
      PChlide
      protochlorophyllide
      BChlide
      bacteriochlorophyllide
      LPOR
      NADPH:protochlorophyllide oxidoreductase
      DPOR
      dark-operative protochlorophyllide reductase
      GSB
      green sulfur bacteria
      CAO
      Chl a oxygenase
      FRL
      far-red light
      COR
      Chlide a oxidoreductase
      3HE
      3-hydroxyethyl
      FDBR
      ferredoxin-dependent bilin reductase.
      bilins, corrins (vitamin B12), siroheme, and coenzyme F430. They are all made from a single, extensively branched biosynthetic pathway and are based on the blueprint of a common biosynthetic primogenitor, uroporphyrinogen III (Fig. 1). These different modified tetrapyrroles vary in the nature of their peripheral side chains, the oxidation state of the macrocycle itself, and the centrally chelated metal ion. Perhaps the most distinctive of all is vitamin B12, which contains a ring-contracted macrocycle and also houses upper and lower ligands in order to provide the octahedral geometry to coordinate the cobalt ion.
      Figure thumbnail gr1
      Figure 1Structures of the major modified tetrapyrroles outlined in this review and their structural relationship to the first macrocyclic primogenitor, uroporphyrinogen III. The major modified tetrapyrroles shown surrounding the central uroporphyrinogen III include chlorophyll aP, coenzyme F430, siroheme, cobalamin, biliverdin IXα, heme d1, and heme b. The asymmetrically arranged pyrrole rings in uroporphyrinogen III are named A–D, with the D ring inverted with respect to the other rings. The numbering scheme for the macrocycle is shown for uroporphyrinogen, where positions 1, 2, 5, 7, 10, 12, 15, and 20 are highlighted. In Chls there is a fifth ring, termed ring E, and similarly in F430, there are two extra rings that are termed E and F as shown. For cobalamin (vitamin B12), the side chains are designated (a–f), and these are labeled. The X above the cobalt is a cyanide group in vitamin B12; this position is occupied by either a methyl or adenosyl group in the major biological forms of cobalamin. The shaded boxes surrounding the names of these end-product compounds coordinate with the colors in other pathway figures and in the summary pathway depicted in .
      The differences in the structures of these molecules are reflected in diverse biological functions. Some of the modified tetrapyrroles are involved in very specific processes; for example, heme d1 is required as a prosthetic group only for the cd1 nitrite reductase (
      • Allen J.W.
      • Barker P.D.
      • Daltrop O.
      • Stevens J.M.
      • Tomlinson E.J.
      • Sinha N.
      • Sambongi Y.
      • Ferguson S.J.
      Why isn't “standard” heme good enough for c-type and d1-type cytochromes?.
      ), whereas others, such as heme, are involved in a myriad of distinct biological roles, from sensing to catalysis (
      • Hamza I.
      • Dailey H.A.
      One ring to rule them all: trafficking of heme and heme synthesis intermediates in the metazoans.
      ). A brief description of the roles played by these molecules is outlined below.
      Chls and the related bacteriochlorophylls (BChls) are the molecules that not only give plants their green pigmentation but are intricately involved in the process of photosynthesis (
      • Barber J.
      Photosynthetic generation of oxygen.
      ). Chls play two roles in photosynthesis: first, they act as antenna molecules and harvest solar energy, and second, they transfer this energy to the reaction centers, where photochemistry occurs that results in the splitting of water or the production of strong reductants for carbon dioxide fixation and ATP generation. With only a few exceptions in which Zn2+ replaces Mg2+, Chls are Mg2+-containing chlorins, and the electronic properties of the chlorin ring allow for the efficient formation of a singlet excited state upon visible light absorption. In contrast to the metal ions found in other modified tetrapyrroles, Mg2+ is not redox-active, and the metal does not play a direct role in the light-trapping process. However, the metal does appear to help potentiate the chemistry of the chlorin ring to make energy transfer more efficient.
      Heme is technically an Fe-containing porphyrin. It has one more double bond in the macrocycle than Chls, and the extra conjugation helps produce the red color associated with the molecule. The central Fe ion is crucial to the functions for which heme is used (
      • Dailey H.A.
      • Dailey T.A.
      • Gerdes S.
      • Jahn D.
      • Jahn M.
      • O'Brian M.R.
      • Warren M.J.
      Prokaryotic heme biosynthesis: multiple pathways to a common essential product.
      ). Iron exists in several oxidation states, and, for this reason, heme has evolved a broad range of roles within biological systems, from acting as a one-electron carrier in respiratory cytochromes to a sensing role for a range of diatomic gases, including CO, NO, and O2. Heme also acts as the prosthetic group in a range of enzymes, including catalases, peroxidases, and cytochromes P450, and is known to be associated with certain transporters and transcription factors (
      • Hamza I.
      • Dailey H.A.
      One ring to rule them all: trafficking of heme and heme synthesis intermediates in the metazoans.
      ).
      The corrinoids, sometimes also referred to as cobamides, encompass cofactors and coenzymes that harbor cobalt-containing, ring-contracted corrin macrocycles. In biologically active corrinoids, the cobalt atom is generally found covalently linked to either a methyl or adenosyl group on the upper face of the macrocycle. The corrin ring is also attached to a lower nucleotide loop via one of its propionate side chains. The nature of the base in this nucleotide loop varies among bacteria, and over 20 different bases are known to be incorporated into corrinoids. The base is specifically dimethylbenzimidazole in vitamin B12, and this specific corrinoid appears to be the only form utilized by eukaryotes. Corrinoids appear to be involved in an ever-increasing number of roles (
      • Bridwell-Rabb J.
      • Drennan C.L.
      Vitamin B12 in the spotlight again.
      ). Methylcorrinoids act as the coenzyme in methyl transfer reactions, such as those mediated by methionine synthase (
      • Banerjee R.
      • Ragsdale S.W.
      The many faces of vitamin B12: catalysis by cobalamin-dependent enzymes.
      ). Adenosylcorrinoids act as the coenzyme in a number of different rearrangement reactions that are mediated by an adenosyl radical, formed from the homolytic cleavage of the Co-adenosyl bond. These reactions include, among many others, methylmalonyl-CoA mutase, ribonucleotide reductase, and the diol dehydratases (
      • Banerjee R.
      • Ragsdale S.W.
      The many faces of vitamin B12: catalysis by cobalamin-dependent enzymes.
      ). Adenosylcobalamin has also recently been shown to be involved as a light sensor in a transcription factor (
      • Jost M.
      • Simpson J.H.
      • Drennan C.L.
      The transcription factor CarH safeguards use of adenosylcobalamin as a light sensor by altering the photolysis products.
      ). Corrinoids without an upper ligand act as the catalytic center for reductive dehalogenases, in which the cobalt ion is thought to form a direct bond with the halide component of the substrate in order to mediate its abstraction (
      • Payne K.A.
      • Quezada C.P.
      • Fisher K.
      • Dunstan M.S.
      • Collins F.A.
      • Sjuts H.
      • Levy C.
      • Hay S.
      • Rigby S.E.
      • Leys D.
      Reductive dehalogenase structure suggests a mechanism for B12 dependent dehalogenation.
      ). Finally, both methylcorrinoids and adenosylcorrinoids also appear to be involved in a specific group of radical SAM enzymes (
      • Bridwell-Rabb J.
      • Drennan C.L.
      Vitamin B12 in the spotlight again.
      ); B12-radical SAM enzymes are the largest group within this broad enzyme class (
      • Wang S.C.
      Cobalamin-dependent radical S-adenosyl-l-methionine enzymes in natural product biosynthesis.
      ).
      Coenzyme F430 is a nickel-containing tetrahydroporphyrinogen and acts as a coenzyme in both forward and reverse methanogenesis (
      • Ragsdale S.W.
      Biochemistry of methyl-coenzyme M reductase: the nickel metalloenzyme that catalyzes the final step in synthesis and the first step in anaerobic oxidation of the greenhouse gas methane.
      ). As a coenzyme within coenzyme M reductase, the central nickel ion is able to mediate the reversible reduction/oxidation of a methyl group to produce methane in both the processes of methanogenesis and anaerobic methane oxidation. In some respects, the binding of nickel in coenzyme F430 mirrors the binding of cobalt in corrins, reflecting similarities in their respective catalytic activities in forming metal-carbon bonds. However, in coenzyme F430, nickel promotes methyl group reduction, whereas in corrinoid-dependent methyltransferases, the cobalt promotes methyl group transfer (
      • Thauer R.K.
      Biochemistry of methanogenesis: a tribute to Marjory Stephenson. 1998 Marjory Stephenson Prize Lecture.
      ). Demonstrating the importance of these tetrapyrrole catalysts, the process of methanogenesis is responsible for the overall production of around 1 billion tons of methane gas per year (
      • Thauer R.K.
      Biochemistry of methanogenesis: a tribute to Marjory Stephenson. 1998 Marjory Stephenson Prize Lecture.
      ).
      The final two modified cyclic tetrapyrroles are siroheme and heme d1. Siroheme is the simplest of the modified tetrapyrroles, and is an Fe-containing isobacteriochlorin (
      • Crane B.R.
      • Getzoff E.D.
      The relationship between structure and function for the sulfite reductases.
      ). It is found mainly in sulfite reductases but also some assimilatory nitrite reductases. The prosthetic group assists in the 6-electron reduction of both sulfite and nitrite to allow their incorporation into biological systems at the level of sulfide and ammonia. It has been suggested that siroheme, in preference to heme, allows a more direct charge transfer route to the active center of these enzymes during the catalytic cycle (
      • Branzanic A.M.V.
      • Ryde U.
      • Silaghi-Dumitrescu R.
      Why does sulfite reductase employ siroheme?.
      ). In contrast, heme d1, which like siroheme is not a heme but a dioxo-isobacteriochlorin, is only utilized by one enzyme, a dissimilatory nitrite reductase called cytochrome cd1.
      Most bilins are derived from heme by oxidative cleavage of the macrocycle to produce biliverdin with release of CO (
      • Wilks A.
      • Heinzl G.
      Heme oxygenation and the widening paradigm of heme degradation.
      ,
      • Wilks A.
      • Ikeda-Saito M.
      Heme utilization by pathogenic bacteria: not all pathways lead to biliverdin.
      ). Ferredoxin-dependent bilin reductases as well as isomerases then lead to the production of four principal bilin pigments: phycocyanobilin, phycoerythrobilin, phycoviolobilin, and phycourobilin. Bilins form the chromophores of phycobiliproteins (
      • Glazer A.N.
      Light guides: directional energy transfer in a photosynthetic antenna.
      ,
      • Ledermann B.
      • Aras M.
      NF-D
      Biosynthesis of cyanobacterial light-harvesting pigments and their assembly into phycobiliproteins.
      ) or the light-sensing photosensors of sensor histidine kinases, including phytochromes or cyanobacteriochromes (
      • Fushimi K.
      • Narikawa R.
      Cyanobacteriochromes: photoreceptors covering the entire UV-to-visible spectrum.
      ,
      • Rockwell N.C.
      • Lagarias J.C.
      Phytochrome diversification in cyanobacteria and eukaryotic algae.
      ).
      As stated earlier, all of these modified tetrapyrroles are synthesized along a branched biochemical pathway from the first and only common macrocyclic intermediate, uroporphyrinogen III. In this review, we will deal with the biogenesis of uroporphyrinogen III and then detail how this macrocycle is converted into the various metallo-prosthetic groups that make up this unique but ubiquitous family of essential life pigments.

      Biosynthesis of uroporphyrinogen III

      The common precursor metabolite for the synthesis of uroporphyrinogen III is the amino ketone, 5-aminolevulinic acid (5-ALA) (
      • Warren M.J.
      • Scott A.I.
      Tetrapyrrole assembly and modification into the ligands of biologically functional cofactors.
      ). This C5 intermediate is uniquely used for the biosynthesis of modified tetrapyrroles and is made by one of two routes (Fig. 2). Some organisms make 5-ALA from a decarboxylating condensation between succinyl-CoA and glycine, called the C4 or Shemin pathway, whereas others make 5-ALA from the intact carbon skeleton of glutamate, called the C5 route. We will briefly review these two pathways and describe how 5-ALA is synthesized.
      Figure thumbnail gr2
      Figure 2The two routes for the biosynthesis of 5-ALA and the subsequent biosynthesis of uroporphyrinogen III. The Shemin, or C4, route involves the condensation of glycine and succinyl-CoA and is mediated by the enzyme 5-aminolevulinic acid synthase. The C5 pathway acquires the intact carbon skeleton from glutamate and utilizes glutamyl-tRNA as an intermediate. The glutamyl-tRNA undergoes a reduction by glutamyl-tRNA reductase to give GSA. The final step involves the enzyme GsaM, which rearranges the GSA into 5-ALA. Then two molecules of 5-ALA are condensed into PBG by the action of the enzyme porphobilinogen synthase. Next, four molecules of PBG are deaminated and linked together to give a linear bilane called HMB in a reaction catalyzed by HMB synthase. The final step involves the cyclization and inversion of the terminal D ring to give uroporphyrinogen III. The gray box for uroporphyrinogen III also identifies this central intermediate in Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, Figure 9, Figure 10, Figure 11, Figure 12, Figure 13, Figure 14.

      The C4 pathway

      The C4 or Shemin route was discovered by Shemin and Rittenberg (
      • Shemin D.
      • Rittenberg D.
      The biological utilization of glycine for the synthesis of the protoporphyrin of hemoglobin.
      ), who had previously shown that the nitrogen atom from glycine was incorporated into heme through labeling studies. Subsequently, Shemin's laboratory and that of Albert Neuberger demonstrated independently that succinyl-CoA condensed with glycine to give 5-ALA (
      • Gibson K.D.
      • Laver W.G.
      • Neuberger A.
      Initial stages in the biosynthesis of porphyrins. 2. The formation of δ-aminolaevulic acid from glycine and succinyl-coenzyme A by particles from chicken erythrocytes.
      ,
      • Kikuchi G.
      • Kumar A.
      • Talmage P.
      • Shemin D.
      The enzymatic synthesis of δ-aminolevulinic acid.
      ). The C4 route is mediated by a single enzyme called 5-aminolevulinic acid synthase (AlaS) (Fig. 2). This enzyme is found in some α-proteobacteria and most eukaryotic organisms apart from higher plants. As well as utilizing both succinyl-CoA and glycine as substrates, the enzyme also employs pyridoxal phosphate as a cofactor. The reaction proceeds through the binding of glycine to the pyridoxal phosphate, forming a Schiff base within the active site. Proton abstraction, followed by attachment of the succinyl-CoA, generates 2-amino-3-ketoadipate as a transient intermediate. Loss of CoA and CO2 then leads to release of 5-ALA from the enzyme. The structure of AlaS from Rhodobacter capsulatus was the first to be solved, confirming that the enzyme is a homodimer (
      • Astner I.
      • Schulze J.O.
      • van den Heuvel J.
      • Jahn D.
      • Schubert W.D.
      • Heinz D.W.
      Crystal structure of 5-aminolevulinate synthase, the first enzyme of heme biosynthesis, and its link to XLSA in humans.
      ). The structure provided many molecular details concerning the active-site residues and their roles in catalysis.

      The C5 pathway

      The detection of AlaS in mammalian and some bacterial systems led to a hunt for the enzyme in other organisms and higher plants. However, no activity could be found, and it took some years before labeling studies with glutamate revealed that plants make 5-ALA from glutamate (
      • Beale S.I.
      • Castelfranco P.A.
      14C incorporation from exogenous compounds into δ-aminolevulinic acid by greening cucumber cotyledons.
      ). It was subsequently shown, through a number of elegant experiments, that glutamate is converted into glutamate semialdehyde (GSA) and then into 5-ALA (
      • Huang D.D.
      • Wang W.Y.
      • Gough S.P.
      • Kannangara C.G.
      δ-Aminolevulinic acid-synthesizing enzymes need an RNA moiety for activity.
      ). The conversion of glutamate into GSA was shown to involve several steps. In fact, it is not glutamate itself that is converted into GSA, but rather glutamyl-tRNA, the same species that is used for mRNA translation. The involvement of glutamyl-tRNA represents one of the few examples in which a tRNA species is used in a process other than protein synthesis.
      The enzyme that catalyzes the transformation of glutamyl-tRNA into GSA is called glutamyl-tRNA reductase, which is now known as GtrR. The enzyme requires NADPH and mediates the synthesis of the aldehyde. The structure of the Methanopyrus kandleri enzyme reveals that the homodimeric protein forms a large asymmetric V-shaped molecule, with a number of distinct domains located along a large helix (
      • Moser J.
      • Schubert W.D.
      • Beier V.
      • Bringemeier I.
      • Jahn D.
      • Heinz D.W.
      V-shaped structure of glutamyl-tRNA reductase, the first enzyme of tRNA-dependent tetrapyrrole biosynthesis.
      ). An active-site cysteine residue in the catalytic domain attacks the glutamate-tRNA bond to form an enzyme thioester with the release of the tRNA moiety. Reduction of the thioester bond by NADPH generates GSA (
      • Hennig M.
      • Grimm B.
      • Contestabile R.
      • John R.A.
      • Jansonius J.N.
      Crystal structure of glutamate-1-semialdehyde aminomutase: an α2-dimeric vitamin B6-dependent enzyme with asymmetry in structure and active site reactivity.
      ). The shape of the enzyme permits an interaction with the next enzyme in the pathway, the GSA aminomutase, GsaM (
      • Lüer C.
      • Schauer S.
      • Möbius K.
      • Schulze J.
      • Schubert W.D.
      • Heinz D.W.
      • Jahn D.
      • Moser J.
      Complex formation between glutamyl-tRNA reductase and glutamate-1-semialdehyde 2,1-aminomutase in Escherichia coli during the initial reactions of porphyrin biosynthesis.
      ). The interaction between GtrR and GsaM allows for channeling of the relatively unstable GSA from one active site to the next. GsaM is also a member of the aminotransferase family and is similar to AlaS (
      • Schulze J.O.
      • Schubert W.D.
      • Moser J.
      • Jahn D.
      • Heinz D.W.
      Evolutionary relationship between initial enzymes of tetrapyrrole biosynthesis.
      ). The protein utilizes pyridoxamine-5′-phosphate as a cofactor to facilitate the rearrangement of the amino group to the C5 position of the molecule. The GSA binds to the pyridoxamine to generate 5′-diaminovalerate, which after rearrangement results in the formation of 5-ALA (
      • Ge H.
      • Lv X.
      • Fan J.
      • Gao Y.
      • Teng M.
      • Niu L.
      Crystal structure of glutamate-1-semialdehyde aminotransferase from Bacillus subtilis with bound pyridoxamine-5′-phosphate.
      ).

      Conversion of 5-ALA into uroporphyrinogen III

      The transformation of 5-ALA into uroporphyrinogen III involves the actions of three enzymes (Fig. 2). These steps are common to all organisms that make modified tetrapyrroles, and no alternative route for the synthesis of uroporphyrinogen III has been described. Initially, 5-ALA is acted upon by an enzyme commonly called porphobilinogen synthase (PbgS), but it is also known as ALA dehydratase. The enzyme oversees a Knorr-type condensation reaction, in which two molecules of ALA are condensed to give the pyrrole porphobilinogen (PBG) (
      • Warren M.J.
      • Cooper J.B.
      • Wood S.P.
      • Shoolingin-Jordan P.M.
      Lead poisoning, haem synthesis and 5-aminolaevulinic acid dehydratase.
      ). A significant amount of mechanistic work has shown that the first ALA molecule to bind to the enzyme gives rise to the propionate side chain of the product and that the second incoming molecule is incorporated into the acetic acid side of the molecule (
      • Jordan P.M.
      • Seehra J.S.
      Mechanism of action of 5-aminolevulinic acid dehydratase: stepwise order of addition of the 2 molecules of 5-aminolevulinic acid in the enzymic-synthesis of porphobilinogen.
      ). The structures of a number of PbgS enzymes have been determined by protein crystallization and X-ray diffraction studies. The yeast enzyme was the first to be determined, and the structure revealed that the enzyme exists as a homooctamer (
      • Erskine P.T.
      • Senior N.
      • Awan S.
      • Lambert R.
      • Lewis G.
      • Tickle I.J.
      • Sarwar M.
      • Spencer P.
      • Thomas P.
      • Warren M.J.
      • Shoolingin-Jordan P.M.
      • Wood S.P.
      • Cooper J.B.
      X-ray structure of 5-aminolaevulinate dehydratase, a hybrid aldolase.
      ). Two important lysine residues occur at the active site of the enzyme and bind the two incoming 5-ALA molecules to form Schiff bases (
      • Warren M.J.
      • Cooper J.B.
      • Wood S.P.
      • Shoolingin-Jordan P.M.
      Lead poisoning, haem synthesis and 5-aminolaevulinic acid dehydratase.
      ). The yeast enzyme contains two Zn2+ ions; one of these is catalytically active, whereas the second appears to play a structural role. In other systems, Mg2+ plays a catalytic role, suggesting that the enzymes utilize the metals to act as Lewis acids within the reaction (
      • Jaffe E.K.
      The porphobilinogen synthase family of metalloenzymes.
      ).
      Four molecules of PBG are next polymerized into a linear bilane (Fig. 2), called hydroxymethylbilane (HMB), which involves the deamination of each of the substrates prior to their incorporation into the product in a highly ordered fashion (
      • Warren M.J.
      • Scott A.I.
      Tetrapyrrole assembly and modification into the ligands of biologically functional cofactors.
      ). The enzyme is known as either HMB synthase (HmbS) or PBG deaminase. It is a monomeric enzyme with a molecular mass of around 35 kDa. It was the first enzyme involved in tetrapyrrole biosynthesis to be crystallized and to have its structure determined (
      • Louie G.V.
      • Brownlie P.D.
      • Lambert R.
      • Cooper J.B.
      • Blundell T.L.
      • Wood S.P.
      • Warren M.J.
      • Woodcock S.C.
      • Jordan P.M.
      Structure of porphobilinogen deaminase reveals a flexible multidomain polymerase with a single catalytic site.
      ). The enzyme contains a very unusual cofactor, a dipyrromethane cofactor, which is also constructed from PBG (
      • Awan S.J.
      • Siligardi G.
      • Shoolingin-Jordan P.M.
      • Warren M.J.
      Reconstitution of the holoenzyme form of Escherichia coli porphobilinogen deaminase from apoenzyme with porphobilinogen and preuroporphyrinogen: a study using circular dichroism spectroscopy.
      ,
      • Jordan P.M.
      • Warren M.J.
      Evidence for a dipyrromethane cofactor at the catalytic site of E. coli porphobilinogen deaminase.
      ) and which is unique to tetrapyrrole biosynthesis. The cofactor is attached to cysteine 242 of the Escherichia coli enzyme through a thioether linkage (
      • Jordan P.M.
      • Warren M.J.
      • Williams H.J.
      • Stolowich N.J.
      • Roessner C.A.
      • Grant S.K.
      • Scott A.I.
      Identification of a cysteine residue as the binding site for the dipyrromethane cofactor at the active site of Escherichia coli porphobilinogen deaminase.
      ). The free α position of the cofactor acts as the elongation site for HMB synthesis (
      • Warren M.J.
      • Jordan P.M.
      Investigation into the nature of substrate binding to the dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase.
      ). The first PBG unit enters the active site and is deaminated through the actions of a catalytic aspartic acid residue. The resulting azafulvene then reacts with the dipyrromethane cofactor, in essence to form a tripyrrole. This first binding pyrrole unit ends up as ring A in the final tetrapyrrole macrocycle. This reaction sequence of PBG binding at the active site, deamination, and attachment to the free α position of the growing polypyrrole chain is repeated three more times until a hexapyrrole is formed, adding in rings B, C, and D of the final macrocycle. At this a point, the link between ring A and the dipyrromethane cofactor is hydrolyzed to generate HMB. The structure of the enzyme reveals a flexible active-site cavity that is lined with a number of arginine residues that help to stabilize and hold the growing polypyrrole entity (
      • Louie G.V.
      • Brownlie P.D.
      • Lambert R.
      • Cooper J.B.
      • Blundell T.L.
      • Wood S.P.
      • Warren M.J.
      • Woodcock S.C.
      • Jordan P.M.
      Structure of porphobilinogen deaminase reveals a flexible multidomain polymerase with a single catalytic site.
      ).
      The final step in the synthesis of uroporphyrinogen III is catalyzed by uroporphyrinogen III synthase (UroS) (Fig. 2). This enzyme not only cyclizes the HMB substrate but also inverts ring D of the bilane (
      • Warren M.J.
      • Scott A.I.
      Tetrapyrrole assembly and modification into the ligands of biologically functional cofactors.
      ). In this respect, the first three rings of the uroporphyrinogen III product are arranged with their acetic acid and propionic acid side chains in the same order, whereas this order is reversed for ring D. This generates the only asymmetric isomer of uroporphyrinogen, providing a molecular handle in terms of substrate orientation with regard to future enzymatic steps (
      • Warren M.J.
      • Scott A.I.
      Tetrapyrrole assembly and modification into the ligands of biologically functional cofactors.
      ). Uroporphyrinogen III synthase is a relatively small, monomeric enzyme with a molecular mass of around 25 kDa. A number of different enzyme mechanisms have been proposed for the enzyme to explain how it is able to mediate ring closure and rearrangement. The only one of these mechanisms to stand up to scrutiny is the spiro mechanism, which involves the formation of a cyclic spiro intermediate, allowing ring D to flip over. A chemically synthesized spiro-lactam analog of this intermediate was shown to act as a strong competitive inhibitor (
      • Stark W.M.
      • Hawker C.J.
      • Hart G.J.
      • Philippides A.
      • Petersen P.M.
      • Lewis J.D.
      • Leeper F.J.
      • Battersby A.R.
      Biosynthesis of porphyrins and related macrocycles. 40. Synthesis of a spiro-lactam related to the proposed spiro-intermediate for porphyrin biosynthesis: inhibition of cosynthetase.
      ). This evidence suggests that the reaction does indeed proceed via a spiro intermediate en route to the formation of uroporphyrinogen III. Surprisingly, for what seems like a complicated reaction, there is very little alignment between the amino acid sequences of uroporphyrinogen III synthases, and no essential active site residue has been identified. However, a number of structures for the enzymes have been elucidated, including one with a bound product (
      • Schubert H.L.
      • Phillips J.D.
      • Heroux A.
      • Hill C.P.
      Structure and mechanistic implications of a uroporphyrinogen III synthase-product complex.
      ). Mutagenesis studies have identified a possible active-site tyrosine residue that may be involved in the elimination of the hydroxyl group from HMB (
      • Roessner C.A.
      • Ponnamperuma K.
      • Scott A.I.
      Mutagenesis identifies a conserved tyrosine residue important for the activity of uroporphyrinogen III synthase from Anacystis nidulans.
      ).
      The biosynthesis of uroporphyrinogen III represents the first major branch point in tetrapyrrole biosynthesis (Fig. 3). Methylation of uroporphyrinogen III at positions 2 and 7 gives rise to precorrin-2, a highly unstable dipyrrocorphin that directs metabolism toward siroheme, cobalamin, and coenzyme F430 biogenesis. In contrast, decarboxylation of uroporphyrinogen III directs the intermediate toward protoporphyrin IXα (ProtoIX) and the biosynthesis of heme and Chls (
      • Warren M.J.
      • Scott A.I.
      Tetrapyrrole assembly and modification into the ligands of biologically functional cofactors.
      ). These two branches will be considered separately, although, as will be seen later, heme can made from either ProtoIX or precorrin-2.
      Figure thumbnail gr3
      Figure 3The biosynthesis of siroheme from uroporphyrinogen III. Initially, uroporphyrinogen is methylated at positions C2 and C7 to give precorrin-2 and then undergoes dehydrogenation to give sirohydrochlorin and finally ferrochelation to yield siroheme. The reactions are either mediated by three independent enzymes, such as SirA, -C, and -B, or by two enzymes, such as a uroporphyrinogen methyltransferase (CobA or Met1p)) and a bifunctional dehydrogenase/chelatase (Met8p), or by a single multifunctional enzyme, CysG. The shaded boxes surrounding the names of compounds coordinate with other pathway figures and the summary depiction in .

      The precorrin-2 branch

      The biosynthesis of siroheme

      Siroheme represents the simplest of the modified tetrapyrroles. It is synthesized in just three steps from uroporphyrinogen III (Fig. 3). Initially, uroporphyrinogen III is methylated at positions C2 and C7 of the macrocyclic ring by uroporphyrinogen III methyltransferase, which requires SAM as a methyl donor (
      • Blanche F.
      • Debussche L.
      • Thibaut D.
      • Crouzet J.
      • Cameron B.
      Purification and characterization of S-adenosyl-l-methionine: uroporphyrinogen III methyltransferase from Pseudomonas denitrificans.
      ). It is thought that the C2 position is methylated first, followed by C7. The resulting product, precorrin-2 (
      • Warren M.J.
      • Roessner C.A.
      • Santander P.J.
      • Scott A.I.
      The Escherichia coli cysG gene encodes S-adenosylmethionine-dependent uroporphyrinogen III methylase.
      ), is also an intermediate in the biosynthesis of vitamin B12, heme d1, and coenzyme F430. The term precorrin arises from its first identification as an intermediate in the biosynthesis of the corrin ring, and all intermediates up to the formation of the corrin ring were given the name precorrin-n, where n refers to the number of methyl groups that have been added to the macrocycle (
      • Uzar H.C.
      • Battersby A.R.
      • Carpenter T.A.
      • Leeper F.J.
      Biosynthesis of porphyrins and related macrocycles. 28. Development of a pulse labeling method to determine the C-methylation sequence for vitamin-B12.
      ). Therefore, precorrin-1 would be the intermediate for which a methyl group has only been added to the C2 position.
      A number of structures of uroporphyrinogen III methyltransferases have been determined, showing a bi-lobal, kidney-shaped enzyme with the active site found at the junction of the two domains (
      • Vévodová J.
      • Graham R.M.
      • Raux E.
      • Schubert H.L.
      • Roper D.I.
      • Brindley A.A.
      • Scott A.I.
      • Roessner C.A.
      • Stamford N.P.J.
      • Stroupe M.E.
      • Getzoff E.D.
      • Warren M.J.
      • Wilson K.S.
      Structure/function studies on a S-adenosyl-(l)-methionine-dependent uroporphyrinogen III C methyltransferase (SUMT), a key regulatory enzyme of tetrapyrrole biosynthesis.
      ). The presence of SAH, one of the products of the methyl transfer reaction, is often seen in the active site and helps to pinpoint where the tetrapyrrole substrate is likely to bind. Indeed, a structure of a uroporphyrinogen methyltransferase with uroporphyrinogen III bound has also been determined (
      • Storbeck S.
      • Saha S.
      • Krausze J.
      • Klink B.U.
      • Heinz D.W.
      • Layer G.
      Crystal structure of the heme d1 biosynthesis enzyme NirE in complex with its substrate reveals new insights into the catalytic mechanism of S-adenosyl-l-methionine-dependent uroporphyrinogen III methyltransferases.
      ). A conserved arginine residue may play a role in helping to promote the methylation events. It is assumed that uroporphyrinogen III binds in the correct orientation to allow methylation at the C2 position and that it then disengages, along with SAH, from the enzyme. After reloading with SAM, the precorrin-1 rebinds with the C7 positioned in close proximity to the SAM to allow the second methylation to take place.
      Precorrin-2 is next acted upon by an NAD+-dependent dehydrogenase, which removes two protons and two electrons from the macrocycle, thereby introducing an extra double bond. This forms an isobacteriochlorin that is called sirohydrochlorin (Fig. 3). In some bacteria, the dehydrogenase exists as a single enzyme called SirC (
      • Schubert H.L.
      • Rose R.S.
      • Leech H.K.
      • Brindley A.A.
      • Hill C.P.
      • Rigby S.E.
      • Warren M.J.
      Structure and function of SirC from Bacillus megaterium: a metal-binding precorrin-2 dehydrogenase.
      ). In other systems, the dehydrogenase also has chelatase activity. One such example of a bifunctional dehydrogenase and chelatase is Met8p from yeast (
      • Schubert H.L.
      • Raux E.
      • Brindley A.A.
      • Leech H.K.
      • Wilson K.S.
      • Hill C.P.
      • Warren M.J.
      The structure of Saccharomyces cerevisiae Met8p, a bifunctional dehydrogenase and ferrochelatase.
      ). This enzyme appears to use the same active site for both the dehydrogenase and chelatase activities. For the chelatase reaction, the enzyme has to insert ferrous iron into sirohydrochlorin to produce siroheme. Although the structure of Met8p has been determined, it is not clear how the chelation reaction is catalyzed or what residues are responsible for the process.
      The overall chelation reaction involves the removal of the two protons attached to the pyrrole nitrogens. In contrast to the lack of information available for the chelatase activity of Met8p, single-function chelatases are also known in different bacterial systems. This includes the SirB enzyme (
      • Leech H.K.
      • Raux-Deery E.
      • Heathcote P.
      • Warren M.J.
      Production of cobalamin and sirohaem in Bacillus megaterium: an investigation into the role of the branchpoint chelatases sirohydrochlorin ferrochelatase (SirB) and sirohydrochlorin cobalt chelatase (CbiX).
      ), which is related to the chelatases associated with cobalt insertion into vitamin B12 via the anaerobic route, ferrochelatases associated with heme synthesis, and the nickel chelatase of F430 synthesis (
      • Brindley A.A.
      • Raux E.
      • Leech H.K.
      • Schubert H.L.
      • Warren M.J.
      A story of chelatase evolution: identification and characterization of a small 13–15-kDa “ancestral” cobaltochelatase (CbiXS) in the archaea.
      ). These chelatases are classified as type II chelatases and are generally single-subunit enzymes that do not require ATP for metal insertion (
      • Brindley A.A.
      • Raux E.
      • Leech H.K.
      • Schubert H.L.
      • Warren M.J.
      A story of chelatase evolution: identification and characterization of a small 13–15-kDa “ancestral” cobaltochelatase (CbiXS) in the archaea.
      ). This contrasts to the type I chelatases associated with Mg2+ insertion during Chl synthesis and cobalt insertion along the aerobic B12 pathway. Structural detail on the type II chelatases has helped identify the main catalytic groups at the active site, which include several histidine residues that could be used to facilitate proton abstraction or metal ion binding (
      • Bali S.
      • Rollauer S.
      • Roversi P.
      • Raux-Deery E.
      • Lea S.M.
      • Warren M.J.
      • Ferguson S.J.
      Identification and characterization of the “missing” terminal enzyme for siroheme biosynthesis in α-proteobacteria.
      ,
      • Romão C.V.
      • Ladakis D.
      • Lobo S.A.
      • Carrondo M.A.
      • Brindley A.A.
      • Deery E.
      • Matias P.M.
      • Pickersgill R.W.
      • Saraiva L.M.
      • Warren M.J.
      Evolution in a family of chelatases facilitated by the introduction of active site asymmetry and protein oligomerization.
      ,
      • Schubert H.L.
      • Raux E.
      • Wilson K.S.
      • Warren M.J.
      Common chelatase design in the branched tetrapyrrole pathways of heme and anaerobic cobalamin synthesis.
      ). The insertion of ferrous iron into the sirohydrochlorin macrocycle generates siroheme (Fig. 3).
      In some organisms, such as E. coli and Salmonella enterica, all three steps of siroheme synthesis, the bis-methylation of uroporphyrinogen III, dehydrogenation, and ferrochelation, are found within a single multifunctional enzyme called CysG (Fig. 3) (
      • Warren M.J.
      • Bolt E.L.
      • Roessner C.A.
      • Scott A.I.
      • Spencer J.B.
      • Woodcock S.C.
      Gene dissection demonstrates that the Escherichia coli cysG gene encodes a multifunctional protein.
      ). In essence, CysG represents a fusion between a uroporphyrinogen III methyltransferase and Met8p. The structure of CysG has been determined to show how these different domains are arranged within a dimeric structure (
      • Stroupe M.E.
      • Leech H.K.
      • Daniels D.S.
      • Warren M.J.
      • Getzoff E.D.
      CysG structure reveals tetrapyrrole-binding features and novel regulation of siroheme biosynthesis.
      ). Interestingly, the enzyme was found to be phosphorylated, suggesting that phosphorylation may play a role in regulating the activity of the enzyme.

      The biosynthesis of coenzyme F430

      Coenzyme F430 is a nickel-containing porphinoid that plays an essential role in the reduction of methyl-coenzyme M in the production of methane gas by methanogens. Similarly, this coenzyme is also involved in anaerobic methane oxidation through a reversal of the process, which allows bacterial consortia to utilize methane as a carbon source (
      • Ragsdale S.W.
      Biochemistry of methyl-coenzyme M reductase: the nickel metalloenzyme that catalyzes the final step in synthesis and the first step in anaerobic oxidation of the greenhouse gas methane.
      ). Coenzyme F430 is clearly based on the uroporphyrinogen III template (Fig. 1) but contains two further rings associated with the cyclization of two side chains (rings E and F). The transformation of uroporphyrinogen III into F430 involves methylation of rings A and B, amidation of the a and c side chains, lactam formation of the amidated c side chain, nickel chelation, macrocycle reduction, and cyclohexanone ring formation (Fig. 4).
      Figure thumbnail gr4
      Figure 4The transformation of sirohydrochlorin into coenzyme F430. The steps involved in the biosynthesis of F430 from sirohydrochlorin are outlined. Initially, sirohydrochlorin is chelated with nickel by the enzyme CfbA to give nickel sirohydrochlorin. Next, the two acetic acid side chains on rings A and B, the a and c side chains, are amidated in a reaction catalyzed by CfbB that also requires glutamine and ATP as substrates. This generates nickel sirohydrochlorin a,c-diamide, which acts as the substrate for the reductase system that is catalyzed by CfbC and -D. The reductase removes three double bonds from the macrocycle, which also spontaneously results in the formation of the lactam ring E, thereby generating seco-F430. The final step, mediated by CfbE, results in the formation of the cyclic hexanone ring F in another ATP-requiring process. The shaded box for coenzyme F430 coordinates with other pathway figures and the summary depiction in .
      Early work on the biosynthesis of coenzyme F430 had shown that the macrocycle was derived from precorrin-2 or sirohydrochlorin (
      • Mucha H.
      • Keller E.
      • Weber H.
      • Lingens F.
      • Trosch W.
      Sirohydrochlorin, a precursor of factor-F430 biosynthesis in Methanobacterium thermoautotrophicum.
      ). However, information on the pathway remained scant, although under certain conditions a ring-F open form of F430, called seco-F430, could be isolated (
      • Pfaltz A.
      • Kobelt A.
      • Hüster R.
      • Thauer R.K.
      Biosynthesis of coenzyme F430 in methanogenic bacteria. Identification of 15,17(3)-seco-F430-17(3)-acid as an intermediate.
      ). Incubation of seco-F430 with a crude cell extract and ATP resulted in the formation of F430, suggesting that seco-F430 was the penultimate intermediate in the pathway. Progress on the biosynthesis of F430 biosynthesis came from analysis of the genomes of a number of different methanogens. In many methanogens, the genes encoding the enzymes for F430 synthesis appear to be dispersed randomly in the genome, but in a few cases the genes are functionally clustered. In several methanogens, five genes are found to localize on the genome. They encode a class II chelatase, an amidase, two reductase subunits (which also display similarity to the subunits of protochlorophyllide reductase of Chl biosynthesis), and a protein showing similarity to a ligase, MurF, involved in peptidoglycan synthesis (
      • Moore S.J.
      • Sowa S.T.
      • Schuchardt C.
      • Deery E.
      • Lawrence A.D.
      • Ramos J.V.
      • Billig S.
      • Birkemeyer C.
      • Chivers P.T.
      • Howard M.J.
      • Rigby S.E.
      • Layer G.
      • Warren M.J.
      Elucidation of the biosynthesis of the methane catalyst coenzyme F430.
      ,
      • Zheng K.
      • Ngo P.D.
      • Owens V.L.
      • Yang X.P.
      • Mansoorabadi S.O.
      The biosynthetic pathway of coenzyme F430 in methanogenic and methanotrophic archaea.
      ). The genes in the cluster were given the locus designation cfb, for cofactor F430 biosynthesis.
      Recombinant production of the encoded proteins of the cfb cluster led to the elucidation of the F430 biosynthetic pathways (Fig. 4) (
      • Moore S.J.
      • Sowa S.T.
      • Schuchardt C.
      • Deery E.
      • Lawrence A.D.
      • Ramos J.V.
      • Billig S.
      • Birkemeyer C.
      • Chivers P.T.
      • Howard M.J.
      • Rigby S.E.
      • Layer G.
      • Warren M.J.
      Elucidation of the biosynthesis of the methane catalyst coenzyme F430.
      ,
      • Zheng K.
      • Ngo P.D.
      • Owens V.L.
      • Yang X.P.
      • Mansoorabadi S.O.
      The biosynthetic pathway of coenzyme F430 in methanogenic and methanotrophic archaea.
      ). The first step was shown to be the chelation of Ni2+ with sirohydrochlorin, mediated by CfbA. This enzyme is a small chelatase, which has a subunit molecular mass of around 12 kDa. It forms a homodimer that has a symmetrical active site. CfbA is much more active with cobalt than nickel in vitro, but it has a preference for nickel in vivo, which indicates that metal delivery and availability play important roles in ensuring that the correct metal is inserted into the correct pathway intermediates (
      • Osman D.
      • Martini M.A.
      • Foster A.W.
      • Chen J.
      • Scott A.J.P.
      • Morton R.J.
      • Steed J.W.
      • Lurie-Luke E.
      • Huggins T.G.
      • Lawrence A.D.
      • Deery E.
      • Warren M.J.
      • Chivers P.T.
      • Robinson N.J.
      Bacterial sensors define intracellular free energies for correct enzyme metalation.
      ). The product of the reaction catalyzed by CfbA is Ni-sirohydrochlorin, which then acts as the substrate for the next enzyme in the pathway, CfbB. This enzyme amidates the a and c acetic acid side chains attached to rings A and B and requires glutamine and ATP as substrates. The enzyme is very similar to the amidase found in cobalamin biosynthesis, CobB, which amidates the same two side chains but on the corrin ring. CfbB generates Ni-sirohydrochlorin a,c-diamide, and this product is the substrate for the reductase system, which removes three double bonds from the macrocycle.
      The reduction is mediated by two subunits, CfbC and CfbD. These subunits are similar not only to the reductase system that removes a double bond in Chl biosynthesis, changing the oxidation of the macrocycle from that of a porphyrin to that of a chlorin, but also to the nitrogenase subunits that are involved in nitrogen fixation. CfbC and CfbD both contain Fe-S centers and couple ATP hydrolysis with reduction of the macrocycle. Prolonged incubation of Ni-sirohydrochlorin a,c-diamide with CfbC and CfbD, together with ATP, resulted in a change in the color of the substrate, from purple to yellow, consistent with reduction of the macrocycle. An analysis of the reaction product indicated that not only had reduction taken place but also the lactam ring E had formed. However, when the reaction with the reductase subunits was incubated for shorter periods of time, then ring E was not formed (
      • Moore S.J.
      • Sowa S.T.
      • Schuchardt C.
      • Deery E.
      • Lawrence A.D.
      • Ramos J.V.
      • Billig S.
      • Birkemeyer C.
      • Chivers P.T.
      • Howard M.J.
      • Rigby S.E.
      • Layer G.
      • Warren M.J.
      Elucidation of the biosynthesis of the methane catalyst coenzyme F430.
      ). It may be that the formation of the lactam ring (E) is a spontaneous chemical reaction as a result of the reduction in the macrocycle. The net result, however, is the formation of seco-F430, which is the substrate for the final enzyme in the pathway, CfbE, which has similarity to the peptidoglycan ligase, MurF. In the presence of ATP, CfbE catalyzes the formation of ring F by cyclizing the propionate side chain on ring D to form a cyclohexanone structure. It is presumed that this reaction proceeds via formation of a phosphorylated intermediate to produce coenzyme F430.

      Biosynthesis of vitamin B12

      Vitamin B12, or cyanocobalamin, is the anti-pernicious anemia factor that was first extracted from raw liver. Structurally, it is composed of a cobalt-containing ring-contracted macrocycle called a corrin (
      • Warren M.J.
      • Raux E.
      • Schubert H.L.
      • Escalante-Semerena J.C.
      The biosynthesis of adenosylcobalamin (vitamin B12).
      ). The cobalt ion is held not only by the four pyrrole nitrogen atoms of the macrocycle itself, but also by two further ligands that are found above (the upper or β-ligand) and below (the lower or α-ligand) the plane of the tetrapyrrole ring. In vitamin B12, the upper ligand is a cyano group, but this is actually a consequence of extraction when cobalamin is produced commercially (
      • Martens J.H.
      • Barg H.
      • Warren M.J.
      • Jahn D.
      Microbial production of vitamin B12.
      ). In biological systems, the upper ligand is normally either a methyl or an adenosyl group in methylcobalamin and adenosylcobalamin, respectively (
      • Banerjee R.
      • Ragsdale S.W.
      The many faces of vitamin B12: catalysis by cobalamin-dependent enzymes.
      ). The lower ligand comes from an unusual base called dimethylbenzimidazole, which is part of a nucleotide loop that is attached to the propionate side chain of ring D. Actually, cobalamin is just one member of a broader class of molecules that are referred to as either corrinoids or cobamides. The variation in corrinoid structures relates to the nature of the lower nucleotide loop and in particular the nature of the base. There are around 20 different corrinoid forms (
      • Degnan P.H.
      • Taga M.E.
      • Goodman A.L.
      Vitamin B12 as a modulator of gut microbial ecology.
      ), of which cobalamin is just one, and it appears to be the only member that is utilized in eukaryotes. The corrinoids are unique among the vitamins in that they are made exclusively by bacteria (
      • Roth J.R.
      • Lawrence J.G.
      • Bobik T.A.
      Cobalamin (coenzyme B12): synthesis and biological significance.
      ). The biosynthesis of these molecules did not make the transition to the eukaryotic world, most likely because of the sheer complexity of the process, which involves about 30 steps. The catalytic properties of corrinoids appear to be associated with the ring-contracted nature of the corrin, which not only holds the cobalt tightly but also acts as an entatic state module, whereby its geometric and electronic conditions are adapted for function, in order to promote changes in the oxidation state of the metal ion (
      • Kieninger C.
      • Deery E.
      • Lawrence A.D.
      • Podewitz M.
      • Wurst K.
      • Nemoto-Smith E.
      • Widner F.J.
      • Baker J.A.
      • Jockusch S.
      • Kreutz C.R.
      • Liedl K.R.
      • Gruber K.
      • Warren M.J.
      • Kräutler B.
      The hydrogenobyric acid structure reveals the corrin ligand as an entatic state module empowering B12-cofactors for catalysis.
      ,
      • Widner F.J.
      • Lawrence A.D.
      • Deery E.
      • Heldt D.
      • Frank S.
      • Gruber K.
      • Wurst K.
      • Warren M.J.
      • Kräutler B.
      Total synthesis, structure, and biological activity of adenosylrhodibalamin, the non-natural rhodium homologue of coenzyme B12.
      ).
      There are two similar although genetically distinct pathways for the biosynthesis of cobalamin (Figure 5, Figure 6), which are referred to as the aerobic and anaerobic routes (
      • Blanche F.
      • Thibaut D.
      • Debussche L.
      • Hertle R.
      • Zipfel F.
      • Muller G.
      Parallels and decisive differences in vitamin B12 biosyntheses.
      ). As their names imply, the pathways differ in their requirement for dioxygen, but they also differ further in the timing of cobalt insertion (
      • Warren M.J.
      • Raux E.
      • Schubert H.L.
      • Escalante-Semerena J.C.
      The biosynthesis of adenosylcobalamin (vitamin B12).
      ). The two pathways diverge at precorrin-2 but rejoin at an intermediate called adenosylcobyrinic acid a,c-diamide. The final steps in cobalamin biosynthesis are similar in both pathways. The anaerobic pathway is the more common of the two routes, with the aerobic pathway being largely restricted to members of the α-proteobacteria (
      • Shelton A.N.
      • Seth E.C.
      • Mok K.C.
      • Han A.W.
      • Jackson S.N.
      • Haft D.R.
      • Taga M.E.
      Uneven distribution of cobamide biosynthesis and dependence in bacteria predicted by comparative genomics.
      ).
      Figure thumbnail gr6
      Figure 6The anaerobic biosynthesis of adenosylcobyric acid from uroporphyrinogen III. The aerobic pathway starts with the synthesis of sirohydrochlorin, which is sometimes also referred to as Factor II. Metal insertion at this stage generates cobalt-sirohydrochlorin, which then undergoes a further methylation at C20 to give cobalt-factor III. Ring contraction is mediated by CbiH, which forms a δ-lactone in the generation of cobalt-precorrin-4. Further methylations coupled with lactone ring opening and rearrangement give rise to cobyrinic acid. Amidations together with adenosylation ultimately give rise to the formation of adenosylcobyric acid. The shaded box surrounding uroporphyrinogen III coordinates with other pathway figures and the summary in .
      Figure thumbnail gr5
      Figure 5The aerobic biosynthesis of adenosylcobyrinic acid a,c-diamide from uroporphyrinogen III. The individual steps along the aerobic route for cobalamin synthesis are shown. Initially, uroporphyrinogen III undergoes three methylation steps at C2, C7, and C20, before hydroxylation at the C20 position generates precorrin-3B, a masked pinacol that is primed for ring contraction through rearrangement. The contraction is mediated by CobJ, which also methylates at C17. More methylations, a decarboxylation, and a mutase reaction generate the orange-colored hydrogenobyrinic acid (HBA) intermediate. Cobalt insertion followed by adenosylation and amidation of the side chains generates adenosylcobyrinic acid a,c-diamide, the point where the aerobic and anaerobic (see Figure 6, Figure 7, Figure 8, Figure 9, Figure 10, Figure 11, Figure 12, Figure 13, Figure 14) pathways rejoin. The gray shading surrounding Uroporphyrinogen III coordinates with other pathway figures and the summary in .
      The biosynthesis of cobalamin represents the most complex of all of the pathways for the biogenesis of modified tetrapyrroles. This reflects not only the high degree of modification that takes place on the tetrapyrrole framework but also the need to add both upper and lower ligands to the cobalt. In this respect, cobalamin is much more three-dimensional than the other members of this ring fellowship. Overall, the biogenesis of adenosylcobalamin from uroporphyrinogen III involves the addition of eight SAM-derived methyl groups, ring contraction whereby the methylated C20 carbon is extruded from the macrocycle, six amidations, a decarboxylation, cobalt insertion, aminopropanol attachment, construction and attachment of the lower nucleotide loop, and adenosylation of the cobalt ion (
      • Warren M.J.
      • Raux E.
      • Schubert H.L.
      • Escalante-Semerena J.C.
      The biosynthesis of adenosylcobalamin (vitamin B12).
      ). As mentioned under “The biosynthesis of siroheme,” the intermediates on the pathway are generally referred to as precorrin-n, where n refers to the number of methyl groups that have been added to the macrocycle (
      • Uzar H.C.
      • Battersby A.R.
      • Carpenter T.A.
      • Leeper F.J.
      Biosynthesis of porphyrins and related macrocycles. 28. Development of a pulse labeling method to determine the C-methylation sequence for vitamin-B12.
      ). The aerobic and anaerobic pathways will be discussed separately.

      The aerobic biosynthesis of cobalamin

      The aerobic pathway (Fig. 5) was elucidated in the organism Pseudomonas denitrificans, and genes for cobalamin biosynthesis are generally given the locus tag cob. Uroporphyrinogen III is methylated by CobA at positions 2 and 7 to give precorrin-2 in a reaction that requires SAM as a methyl donor (
      • Blanche F.
      • Debussche L.
      • Thibaut D.
      • Crouzet J.
      • Cameron B.
      Purification and characterization of S-adenosyl-l-methionine: uroporphyrinogen III methyltransferase from Pseudomonas denitrificans.
      ,
      • Cameron B.
      • Briggs K.
      • Pridmore S.
      • Brefort G.
      • Crouzet J.
      Cloning and analysis of genes involved in coenzyme B12 biosynthesis in Pseudomonas denitrificans.
      ). The next enzyme in the pathway, CobI, then methylates at the C20 position to give precorrin-3A (
      • Thibaut D.
      • Couder M.
      • Crouzet J.
      • Debussche L.
      • Cameron B.
      • Blanche F.
      Assay and purification of S-adenosyl-l-methionine:precorrin-2 methyltransferase from Pseudomonas denitrificans.
      ,
      • Warren M.J.
      • Roessner C.A.
      • Ozaki S.
      • Stolowich N.J.
      • Santander P.J.
      • Scott A.I.
      Enzymatic synthesis and structure of precorrin-3, a trimethyldipyrrocorphin intermediate in vitamin B12 biosynthesis.
      ). The following enzyme in the pathway, CobG, is a monooxygenase that requires dioxygen not only to hydroxylate the C20 position, but also to form a γ-lactone with the acetic acid side chain in ring A (
      • Debussche L.
      • Thibaut D.
      • Danzer M.
      • Debu F.
      • Frechet D.
      • Herman F.
      • Blanche F.
      • Vuilhorgne M.
      Biosynthesis of vitamin-B12: structure of precorrin-3b, the trimethylated substrate of the enzyme catalyzing ring contraction.
      ,
      • Schroeder S.
      • Lawrence A.D.
      • Biedendieck R.
      • Rose R.S.
      • Deery E.
      • Graham R.M.
      • McLean K.J.
      • Munro A.W.
      • Rigby S.E.
      • Warren M.J.
      Demonstration that CobG, the monooxygenase associated with the ring contraction process of the aerobic cobalamin (vitamin B12) biosynthetic pathway, contains an Fe-S center and a mononuclear non-heme iron center.
      ,
      • Scott A.I.
      • Roessner C.A.
      • Stolowich N.J.
      • Spencer J.B.
      • Min C.
      • Ozaki S.I.
      Biosynthesis of vitamin B12: discovery of the enzymes for oxidative ring contraction and insertion of the 4th methyl-group.
      ). This generates precorrin-3B, an intermediate containing a masked pinacol that is primed for ring contraction through a rearrangement reaction in the subsequent step. In R. capsulatus, the same reaction is catalyzed by a quite distinct enzyme called CobZ (
      • McGoldrick H.M.
      • Roessner C.A.
      • Raux E.
      • Lawrence A.D.
      • McLean K.J.
      • Munro A.W.
      • Santabarbara S.
      • Rigby S.E.
      • Heathcote P.
      • Scott A.I.
      • Warren M.J.
      Identification and characterization of a novel vitamin B12 (cobalamin) biosynthetic enzyme (CobZ) from Rhodobacter capsulatus, containing flavin, heme, and Fe-S cofactors.
      ). The actual ring-contraction reaction is catalyzed by CobJ, which not only methylates the C17 position in a SAM-dependent fashion, but also contracts the macrocycle to leave the extruded methylated C20 carbon as an acetyl group attached to C1 (
      • Debussche L.
      • Thibaut D.
      • Danzer M.
      • Debu F.
      • Frechet D.
      • Herman F.
      • Blanche F.
      • Vuilhorgne M.
      Biosynthesis of vitamin-B12: structure of precorrin-3b, the trimethylated substrate of the enzyme catalyzing ring contraction.
      ,
      • Scott A.I.
      • Roessner C.A.
      • Stolowich N.J.
      • Spencer J.B.
      • Min C.
      • Ozaki S.I.
      Biosynthesis of vitamin B12: discovery of the enzymes for oxidative ring contraction and insertion of the 4th methyl-group.
      ,
      • Spencer J.B.
      • Stolowich N.J.
      • Roessner C.A.
      • Min C.H.
      • Scott A.I.
      Biosynthesis of vitamin B12: ring contraction is preceded by incorporation of molecular-oxygen into precorrin-3.
      ,
      • Stolowich N.J.
      • Wang J.J.
      • Spencer J.B.
      • Santander P.J.
      • Roessner C.A.
      • Scott A.I.
      Absolute stereochemistry of precorrin-3x and its relevance to the dichotomy of ring contraction mechanism in vitamin B12 biosynthesis.
      ,
      • Thibaut D.
      • Debussche L.
      • Frechet D.
      • Herman F.
      • Vuilhorgne M.
      • Blanche F.
      Biosynthesis of vitamin B12: the structure of factor-IV, the oxidized form of precorrin-4.
      ,
      • Vishwakarma R.A.
      • Balachandran S.
      • Alanine A.I.D.
      • Stamford N.P.J.
      • Kiuchi F.
      • Leeper F.J.
      • Battersby A.R.
      Biosynthesis of porphyrins and related macrocycles. 41. Fate of oxygen-atoms as precorrin-2 carrying 8 labeled carboxyl groups (13C18O2H) is enzymatically converted into cobyrinic acid.
      ). The product of this reaction is precorrin-4, which acts as the substrate for CobM that adds another SAM-derived methyl group to the macrocycle at C11, yielding precorrin-5 (
      • Debussche L.
      • Thibaut D.
      • Cameron B.
      • Crouzet J.
      • Blanche F.
      Biosynthesis of the corrin macrocycle of coenzyme-B12 in Pseudomonas denitrificans.
      ,
      • Min C.H.
      • Atshaves B.P.
      • Roessner C.A.
      • Stolowich N.J.
      • Spencer J.B.
      • Scott A.I.
      Isolation, structure, and genetically engineered synthesis of precorrin-5, the pentamethylated intermediate of vitamin-B12 biosynthesis.
      ). Methylation at C1 by CobF results in the loss of the extruded C20 position and produces precorrin-6A (
      • Thibaut D.
      • Blanche F.
      • Debussche L.
      • Leeper F.J.
      • Battersby A.R.
      Biosynthesis of vitamin B12: structure of precorrin-6x octamethyl ester.
      ,
      • Thibaut D.
      • Debussche L.
      • Blanche F.
      Biosynthesis of vitamin B12: isolation of precorrin-6x, a metal-free precursor of the corrin macrocycle retaining 5 S-adenosylmethionine-derived peripheral methyl-groups.
      ). A reduction of the macrocycle by CobK results in the loss of a double bond in a reaction that requires NADPH as a cofactor and generates precorrin-6B (
      • Gu S.
      • Sushko O.
      • Deery E.
      • Warren M.J.
      • Pickersgill R.W.
      Crystal structure of CobK reveals strand-swapping between Rossmann-fold domains and molecular basis of the reduced precorrin product trap.
      ,
      • Kiuchi F.
      • Thibaut D.
      • Debussche L.
      • Leeper F.J.
      • Blanche F.
      • Battersby A.R.
      Biosynthesis of vitamin-B12: stereochemistry of transfer of a hydride equivalent from NADPH by precorrin-6x reductase.
      ,
      • Thibaut D.
      • Kiuchi F.
      • Debussche L.
      • Leeper F.J.
      • Blanche F.
      • Battersby A.R.
      Biosynthesis of vitamin B12: structure of the ester of a new biosynthetic intermediate, precorrin-6y.
      ,
      • Weaver G.W.
      • Leeper F.J.
      • Battersby A.R.
      • Blanche F.
      • Thibaut D.
      • Debussche L.
      Biosynthesis of vitamin B12: the site of reduction of precorrin-6x.
      ).
      CobL is the next enzyme in the conveyor belt and mediates methylation at C5 and C15 as well as the decarboxylation of the C12 acetic acid side chain (
      • Blanche F.
      • Famechon A.
      • Thibaut D.
      • Debussche L.
      • Cameron B.
      • Crouzet J.
      Biosynthesis of vitamin B12 in Pseudomonas denitrificans: the biosynthetic sequence from precorrin-6y to precorrin-8x is catalyzed by the Cobl gene product.
      ,
      • Deery E.
      • Schroeder S.
      • Lawrence A.D.
      • Taylor S.L.
      • Seyedarabi A.
      • Waterman J.
      • Wilson K.S.
      • Brown D.
      • Geeves M.A.
      • Howard M.J.
      • Pickersgill R.W.
      • Warren M.J.
      An enzyme-trap approach allows isolation of intermediates in cobalamin biosynthesis.
      ,
      • Thibaut D.
      • Kiuchi F.
      • Debussche L.
      • Blanche F.
      • Kodera M.
      • Leeper F.J.
      • Battersby A.R.
      Biosynthesis of vitamin B12: structural studies on precorrin-8x, an octamethylated intermediate and the structure of its stable tautomer.
      ). CobL represents a fusion between two distinct methyltransferases, with the C-terminal domain being responsible for the decarboxylation of the acetic acid side chain at C12 and the methylation at C15. The N-terminal domain performs the subsequent methylation at C5. The net result of these reactions is the synthesis of precorrin-8, which is the substrate for CobH. This enzyme is responsible for the migration of the methyl group from C11 to C12 (
      • Shipman L.W.
      • Li D.
      • Roessner C.A.
      • Scott A.I.
      • Sacchettini J.C.
      Crystal stucture of precorrin-8x methyl mutase.
      ,
      • Thibaut D.
      • Couder M.
      • Famechon A.
      • Debussche L.
      • Cameron B.
      • Crouzet J.
      • Blanche F.
      The final step in the biosynthesis of hydrogenobyrinic acid is catalyzed by the cobH gene product with precorrin-8x as the substrate.
      ). In so doing, it introduces more conjugation into the macrocycle, forming an orange-colored pigment called hydrogenobyrinic acid. With all of the methylations complete, the amidation of some of the side chains then ensues. CobB amidates the a and c side chains to give hydrogenobyrinic acid a,c-diamide in a reaction that requires ATP and glutamine (
      • Debussche L.
      • Thibaut D.
      • Cameron B.
      • Crouzet J.
      • Blanche F.
      Purification and characterization of cobyrinic acid a,c-diamide synthase from Pseudomonas denitrificans.
      ). The next step involves cobalt chelation, in which an enzyme complex formed between CobN, -S, and -T inserts cobalt in an ATP-dependent fashion to give cobyrinic acid a,c-diamide (
      • Blanche F.
      • Cameron B.
      • Couder M.
      • Crouzet J.
      • Debussche L.
      • Thibaut D.
      Cobalt insertion during coenzyme B12 biosynthesis.
      ,
      • Debussche L.
      • Couder M.
      • Thibaut D.
      • Cameron B.
      • Crouzet J.
      • Blanche F.
      Assay, purification, and characterization of cobaltochelatase, a unique complex enzyme catalyzing cobalt insertion in hydrogenobyrinic acid a,c-diamide during coenzyme B12 biosynthesis in Pseudomonas denitrificans.
      ,
      • Lundqvist J.
      • Elmlund D.
      • Heldt D.
      • Deery E.
      • Söderberg C.A.
      • Hansson M.
      • Warren M.
      • Al-Karadaghi S.
      The AAA+ motor complex of subunits CobS and CobT of cobaltochelatase visualized by single particle electron microscopy.
      ). This chelation reaction, mediated by the class I chelatase CobNST, is similar to the magnesium chelatase reaction of Chl synthesis (see below). With cobalt inserted, the upper ligand is next attached. This involves reduction of the cobalt(II) ion to a cobalt(I) species by a flavin-dependent enzyme called CobR (
      • Lawrence A.D.
      • Deery E.
      • McLean K.J.
      • Munro A.W.
      • Pickersgill R.W.
      • Rigby S.E.
      • Warren M.J.
      Identification, characterization, and structure/function analysis of a corrin reductase involved in adenosylcobalamin biosynthesis.
      ,
      • Lawrence A.D.
      • Taylor S.L.
      • Scott A.
      • Rowe M.L.
      • Johnson C.M.
      • Rigby S.E.
      • Geeves M.A.
      • Pickersgill R.W.
      • Howard M.J.
      • Warren M.J.
      FAD binding, cobinamide binding and active site communication in the corrin reductase (CobR).
      ). The Co(I) species acts as a supernucleophile and is very unstable; it quickly reacts with the adenosyltransferase, CobO, to give adenosylcobyrinic acid a,c-diamide (
      • Debussche L.
      • Couder M.
      • Thibaut D.
      • Cameron B.
      • Crouzet J.
      • Blanche F.
      Purification and partial characterization of cob(I)Alamin adenosyltransferase from Pseudomonas denitrificans.
      ). This is the point at which the aerobic and anaerobic pathways rejoin. These steps are reviewed in more detail elsewhere (
      • Battersby A.R.
      How nature builds the pigments of life: the conquest of vitamin B12.
      ,
      • Blanche F.
      • Cameron B.
      • Crouzet J.
      • Debussche L.
      • Thibaut D.
      • Vuilhorgne M.
      • Leeper F.J.
      • Battersby A.R.
      Vitamin B12: how the problem of its biosynthesis was solved.
      ,
      • Scott A.I.
      Discovering nature's diverse pathways to vitamin B12: a 35-year odyssey.
      ).

      The anaerobic pathway

      The anaerobic pathway (Fig. 6) is characterized by the way that the pathway is able to proceed in the absence of oxygen and also by the early insertion of cobalt. In contrast to the aerobic pathway, the genes for the anaerobic biosynthesis of cobalamin are given the locus tag cbi (
      • Roth J.R.
      • Lawrence J.G.
      • Bobik T.A.
      Cobalamin (coenzyme B12): synthesis and biological significance.
      ,
      • Roth J.R.
      • Lawrence J.G.
      • Rubenfield M.
      • Kieffer-Higgins S.
      • Church G.M.
      Characterization of the cobalamin (vitamin B12) biosynthetic genes of Salmonella typhimurium.
      ). As with the aerobic pathway, the pathway initiates with the bismethylation of uroporphyrinogen III to give precorrin-2. Oxidation of the macrocycle through the removal of two protons and two electrons generates sirohydrochlorin, which is also known as factor II (
      • Warren M.J.
      • Bolt E.L.
      • Roessner C.A.
      • Scott A.I.
      • Spencer J.B.
      • Woodcock S.C.
      Gene dissection demonstrates that the Escherichia coli cysG gene encodes a multifunctional protein.
      ,
      • Raux E.
      • Leech H.K.
      • Beck R.
      • Schubert H.L.
      • Santander P.J.
      • Roessner C.A.
      • Scott A.I.
      • Martens J.H.
      • Jahn D.
      • Thermes C.
      • Rambach A.
      • Warren M.J.
      Identification and functional analysis of enzymes required for precorrin-2 dehydrogenation and metal ion insertion in the biosynthesis of sirohaem and cobalamin in Bacillus megaterium.
      ,
      • Spencer J.B.
      • Stolowich N.J.
      • Roessner C.A.
      • Scott A.I.
      The Escherichia coli cysG gene encodes the multifunctional protein, siroheme synthase.
      ). This represents the substrate for cobalt insertion, whereby a class II chelatase called CbiK or CbiX inserts cobalt into the tetrapyrrole, generating cobalt-factor II (
      • Brindley A.A.
      • Raux E.
      • Leech H.K.
      • Schubert H.L.
      • Warren M.J.
      A story of chelatase evolution: identification and characterization of a small 13–15-kDa “ancestral” cobaltochelatase (CbiXS) in the archaea.
      ,
      • Romão C.V.
      • Ladakis D.
      • Lobo S.A.
      • Carrondo M.A.
      • Brindley A.A.
      • Deery E.
      • Matias P.M.
      • Pickersgill R.W.
      • Saraiva L.M.
      • Warren M.J.
      Evolution in a family of chelatases facilitated by the introduction of active site asymmetry and protein oligomerization.
      ,
      • Leech H.K.
      • Raux E.
      • McLean K.J.
      • Munro A.W.
      • Robinson N.J.
      • Borrelly G.P.
      • Malten M.
      • Jahn D.
      • Rigby S.E.
      • Heathcote P.
      • Warren M.J.
      Characterization of the cobaltochelatase CbiXL: evidence for a 4Fe-4S center housed within an MXCXXC motif.
      ,
      • Raux E.
      • Thermes C.
      • Heathcote P.
      • Rambach A.
      • Warren M.J.
      A role for Salmonella typhimurium cbiK in cobalamin (vitamin B12) and siroheme biosynthesis.
      ). CbiL methylates the C20 position of Cobalt Factor II in a SAM-dependent fashion to give cobalt-factor III (
      • Spencer P.
      • Stolowich N.J.
      • Sumner L.W.
      • Scott A.I.
      Definition of the redox states of cobalt-precorrinoids: investigation of the substrate and redox specificity of CbiL from Salmonella typhimurium.
      ). Ring contraction is next afforded by CbiH, which methylates at C17 and forms a δ-lactone on ring A to give cobalt-precorrin-4 (
      • Moore S.J.
      • Biedendieck R.
      • Lawrence A.D.
      • Deery E.
      • Howard M.J.
      • Rigby S.E.
      • Warren M.J.
      Characterization of the enzyme CbiH60 involved in anaerobic ring contraction of the cobalamin (vitamin B12) biosynthetic pathway.
      ,
      • Santander P.J.
      • Roessner C.A.
      • Stolowich N.J.
      • Holderman M.T.
      • Scott A.I.
      How corrinoids are synthesized without oxygen: nature's first pathway to vitamin B12.
      ). CbiF is the next enzyme in the pathway, and, in the presence of SAM, it methylates at C11 to give cobalt-precorrin-5A (
      • Kajiwara Y.
      • Santander P.J.
      • Roessner C.A.
      • Pérez L.M.
      • Scott A.I.
      Genetically engineered synthesis and structural characterization of cobalt-precorrin 5A and -5B, two new intermediates on the anaerobic pathway to vitamin B12: definition of the roles of the CbiF and CbiG enzymes.
      ,
      • Moore S.J.
      • Lawrence A.D.
      • Biedendieck R.
      • Deery E.
      • Frank S.
      • Howard M.J.
      • Rigby S.E.
      • Warren M.J.
      Elucidation of the anaerobic pathway for the corrin component of cobalamin (vitamin B12).
      ,
      • Ozaki S.I.
      • Roessner C.A.
      • Stolowich N.J.
      • Atshaves B.P.
      • Hertle R.
      • Muller G.
      • Scott A.I.
      Multienzyme synthesis and structure of Factor-S3.
      ,
      • Roessner C.A.
      • Warren M.J.
      • Santander P.J.
      • Atshaves B.P.
      • Ozaki S.
      • Stolowich N.J.
      • Iida K.
      • Scott A.I.
      Expression of 9 Salmonella typhimurium enzymes for cobinamide synthesis: identification of the 11-methyl and 20-methyl transferases of corrin biosynthesis.
      ,
      • Schubert H.L.
      • Wilson K.S.
      • Raux E.
      • Woodcock S.C.
      • Warren M.J.
      The X-ray structure of a cobalamin biosynthetic enzyme, cobalt-precorrin-4 methyltransferase.
      ). The δ-lactone ring is broken by the action of CbiG, which gives rise to cobalt-precorrin-5B and releases the extruded C20 carbon as acetaldehyde (
      • Santander P.J.
      • Roessner C.A.
      • Stolowich N.J.
      • Holderman M.T.
      • Scott A.I.
      How corrinoids are synthesized without oxygen: nature's first pathway to vitamin B12.
      ,
      • Kajiwara Y.
      • Santander P.J.
      • Roessner C.A.
      • Pérez L.M.
      • Scott A.I.
      Genetically engineered synthesis and structural characterization of cobalt-precorrin 5A and -5B, two new intermediates on the anaerobic pathway to vitamin B12: definition of the roles of the CbiF and CbiG enzymes.
      ,
      • Moore S.J.
      • Lawrence A.D.
      • Biedendieck R.
      • Deery E.
      • Frank S.
      • Howard M.J.
      • Rigby S.E.
      • Warren M.J.
      Elucidation of the anaerobic pathway for the corrin component of cobalamin (vitamin B12).
      ,
      • Wang J.
      • Stolowich N.J.
      • Santander P.J.
      • Park J.H.
      • Scott A.I.
      Biosynthesis of vitamin B12: concerning the identity of the two-carbon fragment eliminated during anaerobic formation of cobyrinic acid.
      ). CbiD subsequently methylates at C1 to give cobalt-precorrin-6A (
      • Moore S.J.
      • Lawrence A.D.
      • Biedendieck R.
      • Deery E.
      • Frank S.
      • Howard M.J.
      • Rigby S.E.
      • Warren M.J.
      Elucidation of the anaerobic pathway for the corrin component of cobalamin (vitamin B12).
      ,
      • Roessner C.A.
      • Williams H.J.
      • Scott A.I.
      Genetically engineered production of 1-desmethylcobyrinic acid, 1-desmethylcobyrinic acid a,c-diamide, and cobyrinic acid a,c-diamide in Escherichia coli implies a role for CbiD in C-1 methylation in the anaerobic pathway to cobalamin.
      ). Reduction of the macrocycle by CbiJ in the presence of NADH then produces cobalt-precorrin-6B (
      • Moore S.J.
      • Lawrence A.D.
      • Biedendieck R.
      • Deery E.
      • Frank S.
      • Howard M.J.
      • Rigby S.E.
      • Warren M.J.
      Elucidation of the anaerobic pathway for the corrin component of cobalamin (vitamin B12).
      ). Decarboxylation of the acetic acid side chain attached to C12 and methylation at C15 produces cobalt-precorrin-7 in a reaction mediated by CbiT (
      • Moore S.J.
      • Lawrence A.D.
      • Biedendieck R.
      • Deery E.
      • Frank S.
      • Howard M.J.
      • Rigby S.E.
      • Warren M.J.
      Elucidation of the anaerobic pathway for the corrin component of cobalamin (vitamin B12).
      ,
      • Keller J.P.
      • Smith P.M.
      • Benach J.
      • Christendat D.
      • deTitta G.T.
      • Hunt J.F.
      The crystal structure of MT0146/CbiT suggests that the putative precorrin-8w decarboxylase is a methyltransferase.
      ). A further methylation at C5 by CbiE gives rise to cobyrinic acid (
      • Moore S.J.
      • Lawrence A.D.
      • Biedendieck R.
      • Deery E.
      • Frank S.
      • Howard M.J.
      • Rigby S.E.
      • Warren M.J.
      Elucidation of the anaerobic pathway for the corrin component of cobalamin (vitamin B12).
      ). Amidation of the a and c side chains of the macrocycle by CbiA produces cobyrinic acid a,c-diamide (
      • Fresquet V.
      • Williams L.
      • Raushel F.M.
      Mechanism of cobyrinic acid a,c-diamide synthetase from Salmonella typhimurium LT2.
      ). These steps are reviewed in more detail elsewhere (
      • Moore S.J.
      • Lawrence A.D.
      • Biedendieck R.
      • Deery E.
      • Frank S.
      • Howard M.J.
      • Rigby S.E.
      • Warren M.J.
      Elucidation of the anaerobic pathway for the corrin component of cobalamin (vitamin B12).
      ,
      • Roessner C.A.
      • Scott A.I.
      Fine-tuning our knowledge of the anaerobic route to cobalamin (vitamin B12).
      ).
      The cobyrinic acid a,c-diamide intermediate most likely acts as the substrate for the adenosylation of the cobalt ion by the adenosyltransferase. Within organisms that appear to possess an anaerobic pathway, there seem to be at least three types of adenosyltransferase (
      • Mera P.E.
      • Escalante-Semerena J.C.
      Multiple roles of ATP:cob(I)alamin adenosyltransferases in the conversion of B12 to coenzyme B12.
      ). The first of these is called CobA, which should not be confused with the first methyltransferase of the aerobic pathway (
      • Escalante-Semerena J.C.
      • Suh S.J.
      • Roth J.R.
      cobA function is required for both de novo cobalamin biosynthesis and assimilation of exogenous corrinoids in Salmonella typhimurium.
      ,
      • Maggio-Hall L.A.
      • Escalante-Semerena J.C.
      In vitro synthesis of the nucleotide loop of cobalamin by Salmonella typhimurium enzymes.
      ). This enzyme is orthologous to CobO described in the aerobic pathway above in that it adenosylates the cobalt ion after it has been reduced to a Co(I) species. In the aerobic pathway, the reduction to Co(I) is apparently mediated by free flavin rather than a specific reductase. Two other adenosyltransferases, PduO and EutT, are found within pathways associated with propanediol and ethanolamine utilization, respectively, and are able to substitute for CobA (
      • Buan N.R.
      • Suh S.J.
      • Escalante-Semerena J.C.
      The eutT gene of Salmonella enterica encodes an oxygen-labile, metal-containing ATP:corrinoid adenosyltransferase enzyme.
      ,
      • Mera P.E.
      • St Maurice M.
      • Rayment I.
      • Escalante-Semerena J.C.
      Structural and functional analyses of the human-type corrinoid adenosyltransferase (PduO) from Lactobacillus reuteri.
      ). The processes of either propanediol or ethanolamine utilization take place within specialized proteinaceous organelles called bacterial microcompartments, which both house adenosylcobalamin-dependent enzymes. Within these bacterial microcompartments, PduO and EutT are able to regenerate adenosylcobalamin when the coenzyme periodically becomes occasionally inactivated during the catalytic cycle, and hence these enzymes are not directly involved in de novo biosynthesis (
      • Chowdhury C.
      • Sinha S.
      • Chun S.
      • Yeates T.O.
      • Bobik T.A.
      Diverse bacterial microcompartment organelles.
      ). In the biosynthesis of adenosylcobalamin, the adenosyltransferase results in the synthesis of adenosylcobyrinic acid a,c-diamide.

      Final stages of cobalamin biosynthesis

      After the synthesis of the corrin ring component by either the aerobic or anaerobic pathway, the construction of the final molecule is completed by the synthesis and attachment of the lower nucleotide loop (Fig. 7). The final amidations of the corrin macrocycle are completed by CobQ/CbiP to give adenosylcobyric acid through the addition of four amide groups from glutamine to the b, d, e, and f side chains of the tetrapyrrole framework in an ATP-dependent fashion (
      • Crouzet J.
      • Levy-Schil S.
      • Cameron B.
      • Cauchois L.
      • Rigault S.
      • Rouyez M.C.
      • Blanche F.
      • Debussche L.
      • Thibaut D.
      Nucleotide sequence and genetic analysis of a 13.1-kilobase-pair Pseudomonas denitrificans DNA fragment containing 5 cob genes and identification of structural genes encoding cob(I)alamin adenosyltransferase, cobyric acid synthase, and bifunctional cobinamide kinase-cobinamide phosphate guanylyltransferase.
      ,
      • Williams L.
      • Fresquet V.
      • Santander P.J.
      • Raushel F.M.
      The multiple amidation reactions catalyzed by Cobyric acid synthetase from Salmonella typhimurium are sequential and dissociative.
      ). This gives rise to adenosylcobyric acid. Attachment of an aminopropanol linker to the free carboxylic acid generates adenosylcobinamide in a reaction mediated by CobD/CbiB (
      • Blanche F.
      • Cameron B.
      • Crouzet J.
      • Debussche L.
      • Thibaut D.
      • Vuilhorgne M.
      • Leeper F.J.
      • Battersby A.R.
      Vitamin B12: how the problem of its biosynthesis was solved.
      ,
      • Zayas C.L.
      • Claas K.
      • Escalante-Semerena J.C.
      The CbiB protein of Salmonella enterica is an integral membrane protein involved in the last step of the de novo corrin ring biosynthetic pathway.
      ). The aminopropanol is derived from threonine by the action of either PduX or BluE, which generates threonine phosphate (
      • Fan C.
      • Bobik T.A.
      The PduX enzyme of Salmonella enterica is an l-threonine kinase used for coenzyme B12 synthesis.
      ,
      • Tavares N.K.
      • VanDrisse C.M.
      • Escalante-Semerena J.C.
      Rhodobacterales use a unique l-threonine kinase for the assembly of the nucleotide loop of coenzyme B12.
      ), which is decarboxylated in a pyridoxal phosphate–dependent reaction catalyzed by CobC/CobD to give aminopropanol phosphate (
      • Blanche F.
      • Cameron B.
      • Crouzet J.
      • Debussche L.
      • Thibaut D.
      • Vuilhorgne M.
      • Leeper F.J.
      • Battersby A.R.
      Vitamin B12: how the problem of its biosynthesis was solved.
      ,
      • Brushaber K.R.
      • O'Toole G.A.
      • Escalante-Semerena J.C.
      CobD, a novel enzyme with l-threonine-O-3-phosphate decarboxylase activity, is responsible for the synthesis of (R)-1-amino-2-propanol O-2-phosphate, a proposed new intermediate in cobalamin biosynthesis in Salmonella typhimurium LT2.
      ,
      • Cheong C.G.
      • Bauer C.B.
      • Brushaber K.R.
      • Escalante-Semerena J.C.
      • Rayment I.
      Three-dimensional structure of the l-threonine-O-3-phosphate decarboxylase (CobD) enzyme from Salmonella enterica.
      ). The adenosylcobinamide is primed for the attachment of the lower nucleotide loop by the addition of a GDP moiety, derived from GTP, to the aminopropanol in a reaction catalyzed by the homologous enzymes CobP and CobU of the aerobic and anaerobic pathways, respectively, or the nonhomologous CobY, giving rise to adenosyl-GDP cobinamide (
      • Blanche F.
      • Debussche L.
      • Famechon A.
      • Thibaut D.
      • Cameron B.
      • Crouzet J.
      A bifunctional protein from Pseudomonas denitrificans carries cobinamide kinase and cobinamide phosphate guanylyltransferase activities.
      ,
      • O'Toole G.A.
      • Escalante-Semerena J.C.
      Purification and characterization of the bifunctional CobU enzyme of Salmonella typhimurium LT2. Evidence for a CobU-GMP intermediate.
      ,
      • Woodson J.D.
      • Peck R.F.
      • Krebs M.P.
      • Escalante-Semerena J.C.
      The cobY gene of the archaeon Halobacterium sp. strain NRC-1 is required for de novo cobamide synthesis.
      ).
      Figure thumbnail gr7
      Figure 7The final stages of cobalamin biosynthesis. Adenosylcobyric acid is converted into adenosylcobalamin through the action of three further enzymes. Initially, an aminopropanol phosphate linker is attached to the propionate side chain found on ring D to give adenosylcobinamide phosphate. Aminopropanol is itself derived from threonine. A GDP moiety is attached to the aminopropanol phosphate linker to give adenosyl-GDP-cobinamide. Finally, the GDP moiety is replaced with another nucleotide called α-ribazole, itself made from the ligation of dimethylbenzimidazole with the ribose portion of nicotinamide mononucleotide (NaMN). This results in the formation of adenosylcobalamin. The shaded box surrounding adenosylcobalamin coordinates with other pathway figures and the summary in .
      The unusual dimethylbenzimidazole base is made from either reduced flavin in the presence of oxygen by an enzyme called BluB (
      • Taga M.E.
      • Larsen N.A.
      • Howard-Jones A.R.
      • Walsh C.T.
      • Walker G.C.
      BluB cannibalizes flavin to form the lower ligand of vitamin B12.
      ) or from 5-aminoimidazole ribotide, a branch-point intermediate in thiamine and purine biosynthesis, in four steps (
      • Hazra A.B.
      • Han A.W.
      • Mehta A.P.
      • Mok K.C.
      • Osadchiy V.
      • Begley T.P.
      • Taga M.E.
      Anaerobic biosynthesis of the lower ligand of vitamin B12.
      ), under anoxic conditions. The lower nucleotide is constructed by linking the dimethylbenzimidazole base to nicotinamide mononucleotide in a reaction catalyzed by CobU/T, which generates the α-ribazole phosphate (
      • Cameron B.
      • Blanche F.
      • Rouyez M.C.
      • Bisch D.
      • Famechon A.
      • Couder M.
      • Cauchois L.
      • Thibaut D.
      • Debussche L.
      • Crouzet J.
      Genetic analysis, nucleotide sequence, and products of 2 Pseudomonas denitrificans cob genes encoding nicotinate nucleotide-dimethylbenzimidazole phosphoribosyltransferase and cobalamin (5′-phosphate) synthase.
      ,
      • Cheong C.G.
      • Escalante-Semerena J.C.
      • Rayment I.
      The three-dimensional structures of nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase (CobT) from Salmonella typhimurium complexed with 5,6-dimethybenzimidazole and its reaction products determined to 1.9 Å resolution.
      ,
      • Trzebiatowski J.R.
      • O'Toole G.A.
      • Escalante-Semerena J.C.
      The cobT gene of Salmonella typhimurium encodes the NaMN: 5,6-dimethylbenzimidazole phosphoribosyltransferase responsible for the synthesis of N1-(5-phospho-α-d-ribosyl)-5,6-dimethylbenzimidazole, an intermediate in the synthesis of the nucleotide loop of cobalamin.
      ). The α-ribazole phosphate then displaces the GDP moiety of the adenosyl-GDP cobinamide in a reaction catalyzed by CobV/CobS to generate adenosylcobalamin phosphate (
      • Cameron B.
      • Blanche F.
      • Rouyez M.C.
      • Bisch D.
      • Famechon A.
      • Couder M.
      • Cauchois L.
      • Thibaut D.
      • Debussche L.
      • Crouzet J.
      Genetic analysis, nucleotide sequence, and products of 2 Pseudomonas denitrificans cob genes encoding nicotinate nucleotide-dimethylbenzimidazole phosphoribosyltransferase and cobalamin (5′-phosphate) synthase.
      ,
      • Zayas C.L.
      • Escalante-Semerena J.C.
      Reassessment of the late steps of coenzyme B12 synthesis in Salmonella enterica: evidence that dephosphorylation of adenosylcobalamin-5′-phosphate by the CobC phosphatase is the last step of the pathway.
      ). The phosphate is removed by a phosphatase, CobC, to give adenosylcobalamin (
      • Maggio-Hall L.A.
      • Claas K.R.
      • Escalante-Semerena J.C.
      The last step in coenzyme B12 synthesis is localized to the cell membrane in bacteria and archaea.
      ,
      • O'Toole G.A.
      • Trzebiatowski J.R.
      • Escalante-Semerena J.C.
      The cobC gene of Salmonella typhimurium codes for a novel phosphatase involved in the assembly of the nucleotide loop of cobalamin.
      ). More detailed reviews on the biogenesis of the lower nucleotide loop and the overall synthesis of adenosylcobalamin are found elsewhere (
      • Warren M.J.
      • Raux E.
      • Schubert H.L.
      • Escalante-Semerena J.C.
      The biosynthesis of adenosylcobalamin (vitamin B12).
      ,
      • Blanche F.
      • Cameron B.
      • Crouzet J.
      • Debussche L.
      • Thibaut D.
      • Vuilhorgne M.
      • Leeper F.J.
      • Battersby A.R.
      Vitamin B12: how the problem of its biosynthesis was solved.
      ,
      • Mera P.E.
      • Escalante-Semerena J.C.
      Multiple roles of ATP:cob(I)alamin adenosyltransferases in the conversion of B12 to coenzyme B12.
      ,
      • Mattes T.A.
      • Escalante-Semerena J.C.
      • Deery E.
      • Warren M.J.
      Cobalamin biosynthesis and insertion.
      ).

      Heme biosynthesis: three distinct routes from uroporphyrinogen III

      There was a time, not that long ago, when heme biosynthesis was viewed as something quite straightforward. Now it is recognized that there are three distinct pathways (Fig. 8) for the biosynthesis of heme that are referred to as the protoporphyrin, coproporphyrin, and siroheme pathways (
      • Dailey H.A.
      • Dailey T.A.
      • Gerdes S.
      • Jahn D.
      • Jahn M.
      • O'Brian M.R.
      • Warren M.J.
      Prokaryotic heme biosynthesis: multiple pathways to a common essential product.
      ).
      Figure thumbnail gr8
      Figure 8The three routes to heme from uroporphyrinogen III. The protoporphyrin route (gray arrow) involves the formation of protoporphyrin IX via coproporphyrinogen and protoporphyrinogen with the final step involving insertion of iron into protoporphyrin IX. There are aerobic and anaerobic forms of the enzymes associated with the formation of protoporphyrinogen and protoporphyrin, where the asterisks next to the enzyme (for CgdH, PgdH1, and PgdH2) indicate that these enzymes are found largely under anaerobic conditions. The siroheme route (pale blue arrow) involves the decarboxylation of siroheme to give didecarboxysiroheme, followed by the removal of the acetic acid side chains on rings A and B to give Fe-coproporphyrin before the final step, which involves the decarboxylation of the propionate side chains on rings A and B to produce heme. The coproporphyrin pathway (dusty rose arrow) is a hybrid between the first two routes: coproporphyrinogen is oxidized to give coproporphyrin, which is chelated with iron to give Fe-coproporphyrin. The final step is then the formation of the vinyl side chains through the decarboxylation of the propionate side chains on rings A and B. The conversion of Fe-coproporphyrin into heme is catalyzed by the same enzyme in both the siroheme and coproporphyrin pathways, although it has different names. The shaded boxes surrounding the names of some compounds coordinate with other pathway figures and the summary in .

      The ProtoIX pathway for heme (and Chl) synthesis

      The ProtoIX pathway was long believed to be the only pathway for heme synthesis, involving the transformation of uroporphyrinogen III into ProtoIX in three steps prior to the insertion of iron (Fig. 8). The ProtoIX branch begins with the transformation of uroporphyrinogen III into coproporphyrinogen III through the action of uroporphyrinogen III decarboxylase (
      • Dailey H.A.
      • Dailey T.A.
      • Gerdes S.
      • Jahn D.
      • Jahn M.
      • O'Brian M.R.
      • Warren M.J.
      Prokaryotic heme biosynthesis: multiple pathways to a common essential product.
      ), which decarboxylates the four carboxymethyl side chains attached to the macrocycle with the loss of four molecules of CO2. There is some evidence to suggest that the enzyme is able to mediate the ordered decarboxylation of the carboxymethyl side chains with ring D first followed by A, B, and C. Uroporphyrinogen decarboxylase is able to use the pyrrole rings of the macrocycle to act as an electron sink to help in the catalytic process and hence does not need any exogenous cofactor. The enzyme exists as a homodimer with a subunit molecular mass of around 40 kDa. Structural studies of the free enzyme and the enzyme with bound product have led to the idea that the mechanism may involve conserved arginine and aspartate residues acting as general acids and bases (
      • Phillips J.D.
      • Whitby F.G.
      • Kushner J.P.
      • Hill C.P.
      Structural basis for tetrapyrrole coordination by uroporphyrinogen decarboxylase.
      ,
      • Whitby F.G.
      • Phillips J.D.
      • Kushner J.P.
      • Hill C.P.
      Crystal structure of human uroporphyrinogen decarboxylase.
      ). It has been suggested that uroporphyrinogen decarboxylase is one of the most catalytically proficient enzymes found in nature (
      • Lewis Jr., C.A.
      • Wolfenden R.
      Uroporphyrinogen decarboxylation as a benchmark for the catalytic proficiency of enzymes.
      ).
      Coproporphyrinogen III next undergoes an oxidative decarboxylation of the two propionate (carboxyethyl) side chains attached to rings A and B, to generate vinyl side chains with the release of two more molecules of CO2. The tetrapyrrole product is protoporphyrinogen IX, and the enzyme is now referred to as coproporphyrinogen decarboxylase, CgdC (previously referred to as HemF) (
      • Dailey H.A.
      • Dailey T.A.
      • Gerdes S.
      • Jahn D.
      • Jahn M.
      • O'Brian M.R.
      • Warren M.J.
      Prokaryotic heme biosynthesis: multiple pathways to a common essential product.
      ). The exact mechanism of the enzyme is unknown, and, as with the previous enzyme in the pathway, the reaction does not appear to require any exogenous cofactors or metal ions, although it does require dioxygen (
      • Lash T.D.
      The enigma of coproporphyrinogen oxidase: how does this unusual enzyme carry out oxidative decarboxylations to afford vinyl groups?.
      ). A structure of the oxygen-dependent enzyme has been determined, and the roles of a number of amino acid residues have been investigated (
      • Phillips J.D.
      • Whitby F.G.
      • Warby C.A.
      • Labbe P.
      • Yang C.
      • Pflugrath J.W.
      • Ferrara J.D.
      • Robinson H.
      • Kushner J.P.
      • Hill C.P.
      Crystal structure of the oxygen-depandant coproporphyrinogen oxidase (Hem13p) of Saccharomyces cerevisiae.
      ,
      • Stephenson J.R.
      • Stacey J.A.
      • Morgenthaler J.B.
      • Friesen J.A.
      • Lash T.D.
      • Jones M.A.
      Role of aspartate 400, arginine 262, and arginine 401 in the catalytic mechanism of human coproporphyrinogen oxidase.
      ). Under anoxic conditions, organisms employ an oxygen-independent version of the enzyme, which is technically a coproporphyrinogen dehydrogenase, called CgdH, but which is often referred to as HemN (
      • Layer G.
      • Verfürth K.
      • Mahlitz E.
      • Jahn D.
      Oxygen-independent coproporphyrinogen-III oxidase HemN from Escherichia coli.
      ). This enzyme belongs to the radical SAM superfamily of enzymes. In this case, the enzyme utilizes SAM as a co-substrate to generate an adenosyl radical that participates in the decarboxylation process. HemN was the first radical SAM enzyme to have its structure determined (
      • Layer G.
      • Moser J.
      • Heinz D.W.
      • Jahn D.
      • Schubert W.D.
      Crystal structure of coproporphyrinogen III oxidase reveals cofactor geometry of radical SAM enzymes.
      ). The reaction involves the formation of an adenosyl radical, which is able to abstract a hydrogen from the β-position of the propionate side chain, allowing elimination of the carboxylate as CO2 (
      • Layer G.
      • Pierik A.J.
      • Trost M.
      • Rigby S.E.
      • Leech H.K.
      • Grage K.
      • Breckau D.
      • Astner I.
      • Jänsch L.
      • Heathcote P.
      • Warren M.J.
      • Heinz D.W.
      • Jahn D.
      The substrate radical of Escherichia coli oxygen-independent coproporphyrinogen III oxidase HemN.
      ).
      The final step in the biosynthesis of protoporphyrin IX requires the enzyme protoporphyrinogen oxidase. The enzyme catalyzes the six-electron oxidation of the macrocycle by removing six electrons and six protons with the consequent introduction of three new double bonds. The associated increase in conjugation within the system introduces color, generating the red color associated with porphyrins. As with the previous step in the pathway, there are oxygen-dependent and oxygen-independent versions of the enzyme. The best studied enzyme is oxygen-dependent and was initially named HemY but has been renamed as PgoX (
      • Dailey H.A.
      • Dailey T.A.
      • Gerdes S.
      • Jahn D.
      • Jahn M.
      • O'Brian M.R.
      • Warren M.J.
      Prokaryotic heme biosynthesis: multiple pathways to a common essential product.
      ,
      • Martens J.H.
      • Barg H.
      • Warren M.J.
      • Jahn D.
      Microbial production of vitamin B12.
      ). This is a flavin-dependent enzyme that exists as a homodimer with a subunit molecular mass of around 50 kDa (
      • Dailey H.A.
      • Dailey T.A.
      Protoporphyrinogen oxidase of Myxococcus xanthus: expression, purification, and characterization of the cloned enzyme.
      ). Overall, the reaction requires three molecules of dioxygen, and, because the enzyme contains a tightly but noncovalently bound FAD, it is assumed that the reaction progresses through three two-electron steps, generating three molecules of H2O2 (
      • Dailey H.A.
      • Dailey T.A.
      • Gerdes S.
      • Jahn D.
      • Jahn M.
      • O'Brian M.R.
      • Warren M.J.
      Prokaryotic heme biosynthesis: multiple pathways to a common essential product.
      ). Several structures of PgoX have been determined, although the absence of either a substrate or product complex has precluded any detailed mechanistic proposal (
      • Corradi H.R.
      • Corrigall A.V.
      • Boix E.
      • Mohan C.G.
      • Sturrock E.D.
      • Meissner P.N.
      • Acharya K.R.
      Crystal structure of protoporphyrinogen oxidase from Myxococcus xanthus and its complex with the inhibitor acifluorfen.
      ,
      • Koch M.
      • Breithaupt C.
      • Kiefersauer R.
      • Freigang J.
      • Huber R.
      • Messerschmidt A.
      Crystal structure of protoporphyrinogen IX oxidase: a key enzyme in haem and chlorophyll biosynthesis.
      ). It is also important to note that ProtoIX is an intermediate of the Chl and BChl pathways (see below).
      Two other enzymes that are also able to generate ProtoIX from protoporphyrinogen IX, but only under anoxic conditions, have been identified. Technically, these are protoporphyrinogen dehydrogenases. The first was initially called HemG but has since been renamed PgdH1 (
      • Dailey H.A.
      • Dailey T.A.
      • Gerdes S.
      • Jahn D.
      • Jahn M.
      • O'Brian M.R.
      • Warren M.J.
      Prokaryotic heme biosynthesis: multiple pathways to a common essential product.
      ). This is an FMN-containing enzyme that belongs to the flavodoxin family, and it interacts with the cellular respiratory chain (
      • Boynton T.O.
      • Daugherty L.E.
      • Dailey T.A.
      • Dailey H.A.
      Identification of Escherichia coli HemG as a novel, menadione-dependent flavodoxin with protoporphyrinogen oxidase activity.
      ,
      • Möbius K.
      • Arias-Cartin R.
      • Breckau D.
      • Hannig A.L.
      • Riedmann K.
      • Biedendieck R.
      • Schröder S.
      • Becher D.
      • Magalon A.
      • Moser J.
      • Jahn M.
      • Jahn D.
      Heme biosynthesis is coupled to electron transport chains for energy generation.
      ). The second anaerobic enzyme was initially termed HemJ but has since been renamed PgdH2. This enzyme is the least well-characterized of the ProtoIX-forming systems, as it has not been purified. It is not known whether it is associated with any specific cofactors (
      • Boynton T.O.
      • Gerdes S.
      • Craven S.H.
      • Neidle E.L.
      • Phillips J.D.
      • Dailey H.A.
      Discovery of a gene involved in a third bacterial protoporphyrinogen oxidase activity through comparative genomic analysis and functional complementation.
      ,
      • Kato K.
      • Tanaka R.
      • Sano S.
      • Tanaka A.
      • Hosaka H.
      Identification of a gene essential for protoporphyrinogen IX oxidase activity in the cyanobacterium Synechocystis sp. PCC6803.
      ). However, like PgdH1, it is thought to interact with the respiratory chain.
      Heme is synthesized from ProtoIX by the insertion of ferrous iron into the porphyrin macrocycle with the loss of two protons that were previously attached to the pyrrole nitrogens. The metal-inserting enzyme is a type II chelatase and therefore does not require ATP. This enzyme is often membrane-associated, and a number of them are known to contain an Fe-S cluster, although the function of this redox center is not known (
      • Dailey H.A.
      • Dailey T.A.
      • Wu C.K.
      • Medlock A.E.
      • Wang K.F.
      • Rose J.P.
      • Wang B.C.
      Ferrochelatase at the millennium: structures, mechanisms and [2Fe-2S] clusters.
      ). The best-studied ProtoIX ferrochelatase is the human enzyme, and detailed structural studies have revealed how the substrate is able to bind and induce the associated conformational changes that occur during the catalytic cycle (
      • Medlock A.
      • Swartz L.
      • Dailey T.A.
      • Dailey H.A.
      • Lanzilotta W.N.
      Substrate interactions with human ferrochelatase.
      ).

      The siroheme pathway for heme and heme d1 synthesis

      It has been noted by Sano and co-workers (
      • Ishida T.
      • Yu L.
      • Akutsu H.
      • Ozawa K.
      • Kawanishi S.
      • Seto A.
      • Inubushi T.
      • Sano S.
      A primitive pathway of porphyrin biosynthesis and enzymology in Desulfovibrio vulgaris.
      ) that in sulfate-reducing bacteria, the two methyl groups attached to rings A and B of heme were derived from SAM, strongly indicating that heme was made from precorrin-2. Follow-up research also identified some possible intermediates on the way to heme, but the research remained incomplete. More recently, this new alternative heme pathway was finally elucidated when it was shown that the precursor for heme was actually siroheme (
      • Bali S.
      • Lawrence A.D.
      • Lobo S.A.
      • Saraiva L.M.
      • Golding B.T.
      • Palmer D.J.
      • Howard M.J.
      • Ferguson S.J.
      • Warren M.J.
      Molecular hijacking of siroheme for the synthesis of heme and d1 heme.
      ). Initially, siroheme undergoes a decarboxylation of the acetic acid side chains attached on rings C and D to produce didecarboxysiroheme (Fig. 8). This reaction is mediated by a decarboxylase that is composed of two subunits, AhbA and AhbB. A structure of this enzyme has been solved, revealing how the substrate binds within the active site, in proximity to a number of highly conserved catalytic amino acid residues (
      • Palmer D.J.
      • Schroeder S.
      • Lawrence A.D.
      • Deery E.
      • Lobo S.A.
      • Saraiva L.M.
      • McLean K.J.
      • Munro A.W.
      • Ferguson S.J.
      • Pickersgill R.W.
      • Brown D.G.
      • Warren M.J.
      The structure, function and properties of sirohaem decarboxylase—an enzyme with structural homology to a transcription factor family that is part of the alternative haem biosynthesis pathway.
      ). The next step in the pathway involves a radical SAM enzyme, termed AhbC, which decarboxylates the two carboxymethyl residues on rings A and B. The mechanism underpinning this process has not been elucidated, but the reaction generates Fe-coproporphyrin, which is sometimes referred to as coproheme. The final step in the pathway involves AhbD, another radical SAM enzyme. AhbD carries out a reaction analogous to that catalyzed by CgdH, the coproporphyrinogen dehydrogenase, in that it mediates the decarboxylation of the two propionate side chains attached to rings A and B and their conversion into vinyl side chains. This siroheme pathway for heme biosynthesis is found not only in sulfate-reducing bacteria but also in members of the Archaea (
      • Bali S.
      • Lawrence A.D.
      • Lobo S.A.
      • Saraiva L.M.
      • Golding B.T.
      • Palmer D.J.
      • Howard M.J.
      • Ferguson S.J.
      • Warren M.J.
      Molecular hijacking of siroheme for the synthesis of heme and d1 heme.
      ,
      • Storbeck S.
      • Rolfes S.
      • Raux-Deery E.
      • Warren M.J.
      • Jahn D.
      • Layer G.
      A novel pathway for the biosynthesis of heme in Archaea: genome-based bioinformatic predictions and experimental evidence.
      ). The siroheme pathway is outlined in Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8.
      Significantly, didecarboxysiroheme is also an intermediate for heme d1 synthesis. Heme d1 is an unusual modified tetrapyrrole that is not really a heme at all but is actually a dioxo-isobacteriochlorin (
      • Bali S.
      • Lawrence A.D.
      • Lobo S.A.
      • Saraiva L.M.
      • Golding B.T.
      • Palmer D.J.
      • Howard M.J.
      • Ferguson S.J.
      • Warren M.J.
      Molecular hijacking of siroheme for the synthesis of heme and d1 heme.
      ). The genes associated with the biosynthesis of heme d1 are found within the nir operon in denitrifying bacteria, and include nirD, -L, -G, -H, -F, -J, and -N. As with heme synthesis from siroheme, heme d1 synthesis proceeds via didecarboxysiroheme, in which siroheme is decarboxylated by a combination of NirDL, -G, and -H. The propionic acid side chains attached to rings A and B of the macrocycle are extruded by the action of NirJ (
      • Brindley A.A.
      • Zajicek R.
      • Warren M.J.
      • Ferguson S.J.
      • Rigby S.E.
      NirJ, a radical SAM family member of the d1 heme biogenesis cluster.
      ), a radical SAM enzyme that shares sequence similarity to both AhbC and AhbD. Although NirJ has been purified and has functionally been shown to be a radical SAM enzyme that catalyzes the removal of the two propionate side chains and their replacement with carbonyl groups, its reaction mechanism still has to be elucidated (
      • Boss L.
      • Oehme R.
      • Billig S.
      • Birkemeyer C.
      • Layer G.
      The radical SAM enzyme NirJ catalyzes the removal of two propionate side chains during heme d1 biosynthesis.
      ). The introduction of a double bond into the propionate side chain attached to ring D is mediated by NirN, which uses electron bifurcation to promote the dehydrogenation (
      • Adamczack J.
      • Hoffmann M.
      • Papke U.
      • Haufschildt K.
      • Nicke T.
      • Bröring M.
      • Sezer M.
      • Weimar R.
      • Kuhlmann U.
      • Hildebrandt P.
      • Layer G.
      NirN protein from Pseudomonas aeruginosa is a novel electron-bifurcating dehydrogenase catalyzing the last step of heme d1 biosynthesis.
      ,
      • Klünemann T.
      • Preuss A.
      • Adamczack J.
      • Rosa L.F.M.
      • Harnisch F.
      • Layer G.
      • Blankenfeldt W.
      Crystal structure of dihydro-heme d1 dehydrogenase NirN from Pseudomonas aeruginosa reveals amino acid residues essential for catalysis.
      ).

      The coproporphyrin pathway for heme synthesis

      The third variant of heme biosynthesis was identified after researchers found that some bacteria had a pathway that was not routed through either ProtoIX or siroheme (
      • Dailey H.A.
      • Gerdes S.
      • Dailey T.A.
      • Burch J.S.
      • Phillips J.D.
      Noncanonical coproporphyrin-dependent bacterial heme biosynthesis pathway that does not use protoporphyrin.
      ). In essence, this pathway starts with the decarboxylation of uroporphyrinogen III by UroD to produce coproporphyrinogen as described previously. However, the next step involves the oxidation of the macrocycle to yield coproporphyrin in a reaction that is analogous to the oxidation of protoporphyrinogen to ProtoIX (i.e. the reaction involves the loss of six electrons and six protons) (Fig. 8). The enzyme was first thought to be a protoporphyrinogen oxidase and was initially called HemY, although now it has been renamed CpoX (
      • Dailey H.A.
      • Dailey T.A.
      • Gerdes S.
      • Jahn D.
      • Jahn M.
      • O'Brian M.R.
      • Warren M.J.
      Prokaryotic heme biosynthesis: multiple pathways to a common essential product.
      ). The next step in the pathway involves ferrochelation to give Fe-coproporphyrin or coproheme. The structure of this class II ferrochelatase is very similar to that of ProtoIX ferrochelatase. Evidence suggests that this enzyme works through the distortion of the tetrapyrrole substrate to allow insertion of the metal ion (
      • Al-Karadaghi S.
      • Franco R.
      • Hansson M.
      • Shelnutt J.A.
      • Isaya G.
      • Ferreira G.C.
      Chelatases: distort to select?.
      ). The final step in the pathway involves the decarboxylation of the two propionate side chains on rings A and B to produce the vinyl moieties that are found in heme. This reaction is either catalyzed in some organisms by AhbD or in others by an enzyme called HemQ, which has been renamed ChdC (
      • Dailey H.A.
      • Dailey T.A.
      • Gerdes S.
      • Jahn D.
      • Jahn M.
      • O'Brian M.R.
      • Warren M.J.
      Prokaryotic heme biosynthesis: multiple pathways to a common essential product.
      ). The latter requires hydrogen peroxide as the oxidant, and crystal structures of the enzyme have led to proposed mechanisms involving key catalytic residues within the protein that are positioned adjacent to the relevant propionate side chains (
      • Celis A.I.
      • Gauss G.H.
      • Streit B.R.
      • Shisler K.
      • Moraski G.C.
      • Rodgers K.R.
      • Lukat-Rodgers G.S.
      • Peters J.W.
      • DuBois J.L.
      Structure-based mechanism for oxidative decarboxylation reactions mediated by amino acids and heme propionates in coproheme decarboxylase (HemQ).
      ,
      • Hofbauer S.
      • Mlynek G.
      • Milazzo L.
      • Pühringer D.
      • Maresch D.
      • Schaffner I.
      • Furtmüller P.G.
      • Smulevich G.
      • Djinović-Carugo K.
      • Obinger C.
      Hydrogen peroxide-mediated conversion of coproheme to heme b by HemQ—lessons from the first crystal structure and kinetic studies.
      ). The coproporphyrin pathway is outlined in Fig. 8 and is shown to be a hybrid between the ProtoIX and siroheme routes.

      The transformation of ProtoIX into Chls: Chl a

      Chls are the major absorbers of sunlight on Earth for photosynthesis, and consequently they supply much of the biosphere with energy. The most abundant of these pigments is Chl a, which is found in all oxygenic phototrophs (
      • Björn L.O.
      • Papageorgiou G.C.
      • Blankenship R.E.
      • Govindjee
      A viewpoint: why chlorophyll a?.
      ), but there are also Chls b, c, and d and the recently discovered Chl f (Figure 9, Figure 10) (
      • Chen M.
      • Schliep M.
      • Willows R.D.
      • Cai Z.L.
      • Neilan B.A.
      • Scheer H.
      A red-shifted chlorophyll.
      ). Chls form a structurally and functionally distinct group within the porphyrin family, characterized by the presence of a fifth ring, the isocyclic “E” ring, and an alcohol, usually phytol, esterified at the C173 position. A central magnesium ion is bound via coordinating bonds to the four central nitrogen atoms of the tetrapyrrole. The pyrrole rings of Chls form an extended system of conjugated bonds that confers strong absorption, not only in the blue-violet region of the electromagnetic spectrum, a property shared with many porphyrins, but crucially also in the red. Chls possess other structural features that amplify absorption in the red and also extend it toward 750 nm; these include the carbonyl on ring E, and various side chains at C1–4; the most red-shifted pigment, Chl f, has C2 formyl and C3 vinyl substituents (
      • Chen M.
      • Schliep M.
      • Willows R.D.
      • Cai Z.L.
      • Neilan B.A.
      • Scheer H.
      A red-shifted chlorophyll.
      ). Chls are more than just light absorbers, and when they are situated in an appropriate protein environment, they acquire another function, redox activity, which is crucial for their central role in reaction center complexes (
      • Björn L.O.
      • Papageorgiou G.C.
      • Blankenship R.E.
      • Govindjee
      A viewpoint: why chlorophyll a?.
      ).
      Figure thumbnail gr10
      Figure 10Pathway to show the transformation of chlorophyllide a into other chlorophylls. The addition of the esterifying phytol moieties to the C17 propionates is presumably catalyzed by ChlG in all cases. CAO hydroxylates the C7 methyl group twice, producing a geminal diol that spontaneously dehydrates to form the formyl group of Chl b. The enzyme leading to Chl d in A. marina is unknown. Chl d is also found in some terrestrial cyanobacteria that can photoacclimate to utilize far-red light for oxygenic photosynthesis. There is evidence suggesting that thiol compounds and/or proteins, including cysteine-rich allophycocyanins produced in far-red light, and oxygen may catalyze the formation of Chl d. Note that conversion of the C3 vinyl group of Chl a to the C3 formyl group of Chl d requires the loss of one carbon. The C2 formyl group of Chl f is introduced by a photooxidoreductase, ChlF, which is an enzyme containing Chl a and pheophytin a and which is structurally related to the D1 subunit of photosystem II. The shaded boxes surrounding the names of Chls coordinate with other pathway figures and the summary in .
      Figure thumbnail gr9
      Figure 9The biosynthesis of chlorophyll aP from protoporphyrin IX. Magnesium insertion into ProtoIX directs the intermediate toward Chl synthesis by generating magnesium ProtoIX. This acts as the substrate for a methyltransferase (ChlM), which, together with SAM, gives rise to magnesium ProtoIX monomethyl ester. In the following reaction, the cyclase forms ring E of PChlide a, the C17=C18 double bond of which is then reduced, forming divinyl Chlide a. After reduction of one of the vinyl side chains, geranylgeraniol is attached to the propionate on ring D to form geranylgeranyl Chl a. Subsequent reduction of the geranylgeranyl group to phytol (P) gives rise to Chl aP. The shaded box surrounding Chl aP coordinates with other pathway figures and the summary depiction in .
      The biosynthesis of Chls is initiated when magnesium (Mg2+), rather than the Fe2+ used for heme biosynthesis, is inserted into the ProtoIX macrocycle. The large, multisubunit Mg chelatase complex catalyzes this thermodynamically challenging reaction, in which the free energy of hydrolyzing ∼15 ATP molecules (
      • Adams N.B.
      • Brindley A.A.
      • Hunter C.N.
      • Reid J.D.
      The catalytic power of magnesium chelatase: a benchmark for the AAA+ ATPases.
      ) is used to drive conformational alterations in the complex, likely associated with distortion of the ProtoIX and selective depletion of the hydration shell surrounding the Mg2+. The ∼140-kDa ChlH subunit binds the porphyrin substrate (
      • Karger G.A.
      • Reid J.D.
      • Hunter C.N.
      Characterization of the binding of deuteroporphyrin IX to the magnesium chelatase H subunit and spectroscopic properties of the complex.
      ), and the ∼40-kDa ChlI and ∼80-kDa ChlD subunits belong to the ATPases associated with various cellular activities (AAA+) superfamily. The current view is that ChlD is the physical and mechanical link between the ChlI AAA+ motor and ChlH, which houses the site of metal ion chelation (
      • Farmer D.A.
      • Brindley A.A.
      • Hitchcock A.
      • Jackson P.J.
      • Johnson B.
      • Dickman M.J.
      • Hunter C.N.
      • Reid J.D.
      • Adams N.B.P.
      The ChlD subunit links the motor and porphyrin binding subunits of magnesium chelatase.
      ).
      Although the ChlHID subunits form the catalytic core of magnesium chelatase and are sufficient for in vitro activity, plants and cyanobacteria also require a small (∼26-kDa) soluble protein, Gun4, and make little Chl in its absence (
      • Larkin R.M.
      • Alonso J.M.
      • Ecker J.R.
      • Chory J.
      GUN4, a regulator of chlorophyll synthesis and intracellular signaling.
      ). Gun4 lowers the magnesium concentration required for chelatase activity at low porphyrin concentrations (
      • Davison P.A.
      • Schubert H.L.
      • Reid J.D.
      • Iorg C.D.
      • Heroux A.
      • Hill C.P.
      • Hunter C.N.
      Structural and biochemical characterization of Gun4 suggests a mechanism for its role in chlorophyll biosynthesis.
      ) and substantially enhances the catalytic rate, by at least 10-fold (
      • Adams N.B.
      • Brindley A.A.
      • Hunter C.N.
      • Reid J.D.
      The catalytic power of magnesium chelatase: a benchmark for the AAA+ ATPases.
      ). There are structures of apo-ChlH (
      • Chen X.
      • Pu H.
      • Fang Y.
      • Wang X.
      • Zhao S.
      • Lin Y.
      • Zhang M.
      • Dai H.E.
      • Gong W.
      • Liu L.
      Crystal structure of the catalytic subunit of magnesium chelatase.
      ), apo-Gun4 (
      • Davison P.A.
      • Schubert H.L.
      • Reid J.D.
      • Iorg C.D.
      • Heroux A.
      • Hill C.P.
      • Hunter C.N.
      Structural and biochemical characterization of Gun4 suggests a mechanism for its role in chlorophyll biosynthesis.
      ,
      • Tarahi Tabrizi S.
      • Sawicki A.
      • Zhou S.
      • Luo M.
      • Willows R.D.
      GUN4-protoporphyrin IX is a singlet oxygen generator with consequences for plastid retrograde signaling.
      ,
      • Verdecia M.A.
      • Larkin R.M.
      • Ferrer J.L.
      • Riek R.
      • Chory J.
      • Noel J.P.
      Structure of the Mg-chelatase cofactor GUN4 reveals a novel hand-shaped fold for porphyrin binding.
      ), and Synechocystis sp. PCC 6803 Gun4 bound to both deuteroporphyrin and Mg-deuteroporphyrin (
      • Chen X.
      • Pu H.
      • Wang X.
      • Long W.
      • Lin R.
      • Liu L.
      Crystal structures of GUN4 in complex with porphyrins.
      ); however, structures of subcomplexes, and the entire Mg chelatase complex at various stages of the catalytic cycle, will be required for a complete characterization of this important enzyme complex.
      The next step in Chl biosynthesis involves esterification of the propionate side chain attached to ring C with a methyl group, forming Mg-ProtoIX monomethylester (Fig. 9) (
      • Smith C.A.
      • Suzuki J.Y.
      • Bauer C.E.
      Cloning and characterization of the chlorophyll biosynthesis gene chlM from Synechocystis PCC 6803 by complementation of a bacteriochlorophyll biosynthesis mutant of Rhodobacter capsulatus.
      ). This reaction, which requires SAM as the methyl donor, is catalyzed by the enzyme S-adenosyl-l-methionine Mg-ProtoIX methyltransferase (ChlM) (
      • Shepherd M.
      • Reid J.D.
      • Hunter C.N.
      Purification and kinetic characterization of the magnesium protoporphyrin IX methyltransferase from Synechocystis PCC6803.
      ). Steady-state and transient kinetic analyses of ChlM, produced heterologously in E. coli, show that the reaction proceeds by a random-binding mechanism that forms a ternary ChlM-SAM-Mg-ProtoIX complex (
      • Shepherd M.
      • Hunter C.N.
      Transient kinetics of the reaction catalysed by magnesium protoporphyrin IX methyltransferase.
      ). Intriguingly, the magnesium chelatase subunit ChlH accelerates the formation and breakdown of an intermediate in the catalytic cycle of ChlM (
      • Shepherd M.
      • McLean S.
      • Hunter C.N.
      Kinetic basis for linking the first two enzymes of chlorophyll biosynthesis.
      ). Another link between the two first committed steps of Chl biosynthesis is suggested by Gun4, which could play a role in trafficking Mg-ProtoIX from ChlH to ChlM; the propionate group of Mg-deuteroporphyrin that is methylated by ChlM has been observed to protrude from the binding cleft of Gun4, potentially exposing it to ChlM (
      • Chen X.
      • Wang X.
      • Feng J.
      • Chen Y.
      • Fang Y.
      • Zhao S.
      • Zhao A.
      • Zhang M.
      • Liu L.
      Structural insights into the catalytic mechanism of Synechocystis magnesium protoporphyrin IX O-methyltransferase (ChlM).
      ).
      The methylated propionate side chain on ring C is used to form the isocyclic fifth (E) ring (Fig. 9), in a series of reactions catalyzed by Mg-ProtoIX monomethylester (oxidative) ring cyclase (cyclase) that produce 3,8-divinylprotochlorophyllide (divinyl-PChlide). The formation of ring E is accompanied by a transition to a green color, and this structural change creates an absorption band at 630 nm, a crucial step in the eventual formation of Chl a with strong absorption at 665 nm in methanol. Experiments with 18O-labeled molecular oxygen showed its direct incorporation into the carbonyl group of the isocyclic ring (
      • Walker C.J.
      • Mansfield K.E.
      • Smith K.M.
      • Castelfranco P.A.
      Incorporation of atmospheric oxygen into the carbonyl functionality of the protochlorophyllide isocyclic ring.
      ), so this enzyme is referred to as an oxidative, aerobic, or O2-dependent cyclase. There is also an O2-independent cyclase, BchE, which incorporates oxygen donated from water (
      • Porra R.J.
      • Schäfer W.
      • Gad'on N.
      • Katheder I.
      • Drews G.
      • Scheer H.
      Origin of the two carbonyl oxygens of bacteriochlorophyll a: demonstration of two different pathways for the formation of ring E in Rhodobacter sphaeroides and Roseobacter denitrificans, and a common hydratase mechanism for 3-acetyl group formation.
      ). Although BchE homologs (termed ChlE) have been found in some cyanobacteria (
      • Yamanashi K.
      • Minamizaki K.
      • Fujita Y.
      Identification of the chlE gene encoding oxygen-independent Mg-protoporphyrin IX monomethyl ester cyclase in cyanobacteria.
      ), the O2-independent cyclase is found mainly in anoxygenic phototrophic bacteria, and it will be described under “The biosynthesis of BChls a, b, and g.” Pinta et al. (
      • Pinta V.
      • Picaud M.
      • Reiss-Husson F.
      • Astier C.
      Rubrivivax gelatinosus acsF (previously orf358) codes for a conserved, putative binuclear-iron-cluster-containing protein involved in aerobic oxidative cyclization of Mg-protoporphyrin IX monomethylester.
      ) assigned AcsF (aerobic cyclization system Fe-containing subunit) in the purple betaproteobacterium Rubrivivax gelatinosus to the O2-dependent reaction, and homologs were subsequently found in all oxygenic photosynthetic organisms investigated. These include the genes Crd1 and Cth1 in Chlamydomonas reinhardtii (
      • Moseley J.
      • Quinn J.
      • Eriksson M.
      • Merchant S.
      The Crd1 gene encodes a putative di-iron enzyme required for photosystem I accumulation in copper deficiency and hypoxia in Chlamydomonas reinhardtii.
      ,
      • Moseley J.L.
      • Page M.D.
      • Alder N.P.
      • Eriksson M.
      • Quinn J.
      • Soto F.
      • Theg S.M.
      • Hippler M.
      • Merchant S.
      Reciprocal expression of two candidate di-iron enzymes affecting photosystem I and light-harvesting complex accumulation.
      ), sll1214 and sll1874 in Synechocystis sp. PCC 6803 (
      • Minamizaki K.
      • Mizoguchi T.
      • Goto T.
      • Tamiaki H.
      • Fujita Y.
      Identification of two homologous genes, chlAI and chlAII, that are differentially involved in isocyclic ring formation of chlorophyll a in the cyanobacterium Synechocystis sp. PCC 6803.
      ,
      • Peter E.
      • Salinas A.
      • Wallner T.
      • Jeske D.
      • Dienst D.
      • Wilde A.
      • Grimm B.
      Differential requirement of two homologous proteins encoded by sll1214 and sll1874 for the reaction of Mg protoporphyrin monomethylester oxidative cyclase under aerobic and micro-oxic growth conditions.
      ), Chl27 in Arabidopsis (
      • Tottey S.
      • Block M.A.
      • Allen M.
      • Westergren T.
      • Albrieux C.
      • Scheller H.V.
      • Merchant S.
      • Jensen P.E.
      Arabidopsis CHL27, located in both envelope and thylakoid membranes, is required for the synthesis of protochlorophyllide.
      ), and Xantha-I in barley (
      • Rzeznicka K.
      • Walker C.J.
      • Westergren T.
      • Kannangara C.G.
      • von Wettstein D.
      • Merchant S.
      • Gough S.P.
      • Hansson M.
      Xantha-l encodes a membrane subunit of the aerobic Mg-protoporphyrin IX monomethyl ester cyclase involved in chlorophyll biosynthesis.
      ). Three classes of O2-dependent cyclases have been identified: in betaproteobacteria, AcsF is sufficient, whereas oxygenic phototrophs require an auxiliary subunit, Ycf54 (
      • Hollingshead S.
      • Kopecná J.
      • Jackson P.J.
      • Canniffe D.P.
      • Davison P.A.
      • Dickman M.J.
      • Sobotka R.
      • Hunter C.N.
      Conserved chloroplast open-reading frame ycf54 is required for activity of the magnesium protoporphyrin monomethylester oxidative cyclase in Synechocystis PCC 6803.