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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.
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717
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.
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” (
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.
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 (
). 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 (
). 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 (
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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
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) (
). 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.
The C4 pathway
The C4 or Shemin route was discovered by Shemin and Rittenberg (
), 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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
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) (
). 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 (
). 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 (
). 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 (
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 (
). 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 (
). 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 (
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 (
). 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 (
). 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 (
). 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 (
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 (
). These two branches will be considered separately, although, as will be seen later, heme can made from either ProtoIX or precorrin-2.
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 (
), 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 (
). 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 (
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 (
). 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 (
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 (
). 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 (
). 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) (
). 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 (
). 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 (
). 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).
Early work on the biosynthesis of coenzyme F430 had shown that the macrocycle was derived from precorrin-2 or sirohydrochlorin (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
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 (
). 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 (
). 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 (
). 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 (
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.
). 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 (
). 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 (
). 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 (
). 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 (
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.
). 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 (
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 (
). 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 (
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.
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.
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.
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 (
). 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 (
). 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 (
). 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 (
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.
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.
). 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 (
), 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 (
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.
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.
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.
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 (
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 (
), 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 (
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) (
). 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 (
). 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 (
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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
) 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 (
). 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 (
). 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 (
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 (
). 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 (
), 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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
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 (
) 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 (
), 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 (
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 (
). 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 (
). 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 (
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 (
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.
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 (
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.