Enzymology
A structure-function analysis of chlorophyllase reveals a mechanism for activity regulation dependent on disulfide bonds
Chlorophyll pigments are used by photosynthetic organisms to facilitate light capture and mediate the conversion of sunlight into chemical energy. Due to the indispensable nature of this pigment and its propensity to form reactive oxygen species, organisms heavily invest in its biosynthesis, recycling, and degradation. One key enzyme implicated in these processes is chlorophyllase, an α/β hydrolase that hydrolyzes the phytol tail of chlorophyll pigments to produce chlorophyllide molecules. This enzyme was discovered a century ago, but despite its importance to diverse photosynthetic organisms, there are still many missing biochemical details regarding how chlorophyllase functions.Structures of the NDP-pyranose mutase belonging to glycosyltransferase family 75 reveal residues important for Mn2+ coordination and substrate binding
Members of glycosyltransferase family 75 (GT75) not only reversibly catalyze the autoglycosylation of a conserved arginine residue with specific NDP-sugars but also exhibit NDP-pyranose mutase activity that reversibly converts specific NDP-pyranose to NDP-furanose. The latter activity provides valuable NDP-furanosyl donors for glycosyltransferases and requires a divalent cation as a cofactor instead of FAD used by UDP-D-galactopyranose mutase. However, details of the mechanism for NDP-pyranose mutase activity are not clear.Crystal structures of Schistosoma mansoni histone deacetylase 8 reveal a novel binding site for allosteric inhibitors
Parasitic diseases cause significant global morbidity and mortality particularly in the poorest regions of the world. Schistosomiasis, one of the most widespread neglected tropical diseases, affects more than 200 million people worldwide. Histone deacetylase (HDAC) inhibitors are prominent epigenetic drugs that are being investigated in the treatment of several diseases, including cancers and parasitic diseases. Schistosoma mansoni HDAC8 (SmHDAC8) is highly expressed in all life cycle stages of the parasite, and selective inhibition is required in order to avoid undesirable off-target effects in the host.Crystal structure of mevalonate 3,5-bisphosphate decarboxylase reveals insight into the evolution of decarboxylases in the mevalonate metabolic pathways
Mevalonate 3,5-bisphosphate decarboxylase is involved in the recently discovered Thermoplasma-type mevalonate pathway. The enzyme catalyzes the elimination of the 3-phosphate group from mevalonate 3,5-bisphosphate as well as concomitant decarboxylation of the substrate. This entire reaction of the enzyme resembles the latter half-reactions of its homologs, diphosphomevalonate decarboxylase and phosphomevalonate decarboxylase, which also catalyze ATP-dependent phosphorylation of the 3-hydroxyl group of their substrates.Crystal structure of Grimontia hollisae collagenase provides insights into its novel substrate specificity toward collagen
Collagenase from the gram-negative bacterium Grimontia hollisae strain 1706B (Ghcol) degrades collagen more efficiently even than clostridial collagenase, the most widely used industrial collagenase. However, the structural determinants facilitating this efficiency are unclear. Here, we report the crystal structures of ligand-free and Gly-Pro-hydroxyproline (Hyp)-complexed Ghcol at 2.2 and 2.4 Å resolution, respectively. These structures revealed that the activator and peptidase domains in Ghcol form a saddle-shaped structure with one zinc ion and four calcium ions.Structural and biochemical characterization of the prenylated flavin mononucleotide-dependent indole-3-carboxylic acid decarboxylase
The ubiquitous UbiD family of reversible decarboxylases is implicated in a wide range of microbial processes and depends on the prenylated flavin mononucleotide cofactor for catalysis. However, only a handful of UbiD family members have been characterized in detail, and comparison between these has suggested considerable variability in enzyme dynamics and mechanism linked to substrate specificity. In this study, we provide structural and biochemical insights into the indole-3-carboxylic acid decarboxylase, representing an UbiD enzyme activity distinct from those previously studied.Structural diversity and substrate preferences of three tannase enzymes encoded by the anaerobic bacterium Clostridium butyricum
Tannins are secondary metabolites that are enriched in the bark, roots, and knots in trees and are known to hinder microbial attack. The biological degradation of water-soluble gallotannins, such as tannic acid, is initiated by tannase enzymes (EC 3.1.1.20), which are esterases able to liberate gallic acid from aromatic-sugar complexes. However, only few tannases have previously been studied in detail. Here, for the first time, we biochemically and structurally characterize three tannases from a single organism, the anaerobic bacterium Clostridium butyricum, which inhabits both soil and gut environments.Engineering of tissue inhibitor of metalloproteinases TIMP-1 for fine discrimination between closely related stromelysins MMP-3 and MMP-10
Matrix metalloproteinases (MMPs) have long been known as key drivers in the development and progression of diseases, including cancer and neurodegenerative, cardiovascular, and many other inflammatory and degenerative diseases, making them attractive potential drug targets. Engineering selective inhibitors based upon tissue inhibitors of metalloproteinases (TIMPs), endogenous human proteins that tightly yet nonspecifically bind to the family of MMPs, represents a promising new avenue for therapeutic development.Characterization of a lytic polysaccharide monooxygenase from Aspergillus fumigatus shows functional variation among family AA11 fungal LPMOs
The discovery of oxidative cleavage of recalcitrant polysaccharides by lytic polysaccharide monooxygenases (LPMOs) has affected the study and industrial application of enzymatic biomass processing. Despite being widespread in fungi, LPMOs belonging to the auxiliary activity (AA) family AA11 have been understudied. While these LPMOs are considered chitin active, some family members have little or no activity toward chitin, and the only available crystal structure of an AA11 LPMO lacks features found in bacterial chitin-active AA10 LPMOs.Structure of a bacterial α-1,2-glucosidase defines mechanisms of hydrolysis and substrate specificity in GH65 family hydrolases
Glycoside hydrolase family 65 (GH65) comprises glycoside hydrolases (GHs) and glycoside phosphorylases (GPs) that act on α-glucosidic linkages in oligosaccharides. All previously reported bacterial GH65 enzymes are GPs, whereas all eukaryotic GH65 enzymes known are GHs. In addition, to date, no crystal structure of a GH65 GH has yet been reported. In this study, we use biochemical experiments and X-ray crystallography to examine the function and structure of a GH65 enzyme from Flavobacterium johnsoniae (FjGH65A) that shows low amino acid sequence homology to reported GH65 enzymes.Patient mutations in human ATP:cob(I)alamin adenosyltransferase differentially affect its catalytic versus chaperone functions
Human ATP:cob(I)alamin adenosyltransferase (ATR) is a mitochondrial enzyme that catalyzes an adenosyl transfer to cob(I)alamin, synthesizing 5′-deoxyadenosylcobalamin (AdoCbl) or coenzyme B12. ATR is also a chaperone that escorts AdoCbl, transferring it to methylmalonyl-CoA mutase, which is important in propionate metabolism. Mutations in ATR lead to methylmalonic aciduria type B, an inborn error of B12 metabolism. Our previous studies have furnished insights into how ATR protein dynamics influence redox-linked cobalt coordination chemistry, controlling its catalytic versus chaperone functions.Identification of difructose dianhydride I synthase/hydrolase from an oral bacterium establishes a novel glycoside hydrolase family
Fructooligosaccharides and their anhydrides are widely used as health-promoting foods and prebiotics. Various enzymes acting on β-D-fructofuranosyl linkages of natural fructan polymers have been used to produce functional compounds. However, enzymes that hydrolyze and form α-D-fructofuranosyl linkages have been less studied. Here, we identified the BBDE_2040 gene product from Bifidobacterium dentium (α-D-fructofuranosidase and difructose dianhydride I synthase/hydrolase from Bifidobacterium dentium [αFFase1]) as an enzyme with α-D-fructofuranosidase and α-D-arabinofuranosidase activities and an anomer-retaining manner.Catalytic and structural insights into a stereospecific and thermostable Class II aldolase HpaI from Acinetobacter baumannii
Aldolases catalyze the reversible reactions of aldol condensation and cleavage and have strong potential for the synthesis of chiral compounds, widely used in pharmaceuticals. Here, we investigated a new Class II metal aldolase from the p-hydroxyphenylacetate degradation pathway in Acinetobacter baumannii, 4-hydroxy-2-keto-heptane-1,7-dioate aldolase (AbHpaI), which has various properties suitable for biocatalysis, including stereoselectivity/stereospecificity, broad aldehyde utilization, thermostability, and solvent tolerance.Structural and biochemical insights into CRISPR RNA processing by the Cas5c ribonuclease SMU1763 from Streptococcus mutans
The cariogenic pathogen Streptococcus mutans contains two CRISPR systems (type I-C and type II-A) with the Cas5c protein (SmuCas5c) involved in processing of long CRISPR RNA transcripts (pre-crRNA) containing repeats and spacers to mature crRNA guides. In this study, we determined the crystal structure of SmuCas5c at a resolution of 1.72 Å, which revealed the presence of an N-terminal modified RNA recognition motif and a C-terminal twisted β-sheet domain with four bound sulphate molecules. Analysis of surface charge and residue conservation of the SmuCas5c structure suggested the location of an RNA-binding site in a shallow groove formed by the RNA recognition motif domain with several conserved positively charged residues (Arg39, Lys52, Arg109, Arg127, and Arg134).In silico and in vitro analysis of an Aspergillus niger chitin deacetylase to decipher its subsite sugar preferences
Chitin deacetylases (CDAs) are found in many different organisms ranging from marine bacteria to fungi and insects. These enzymes catalyze the removal of acetyl groups from chitinous substrates generating various chitosans, linear copolymers consisting of N-acetylglucosamine (GlcNAc) and glucosamine. CDAs influence the degree of acetylation of chitosans as well as their pattern of acetylation, a parameter that was recently shown to influence the physicochemical properties and biological activities of chitosans.N-acetylmannosamine-6-phosphate 2-epimerase uses a novel substrate-assisted mechanism to catalyze amino sugar epimerization
There are five known general catalytic mechanisms used by enzymes to catalyze carbohydrate epimerization. The amino sugar epimerase N-acetylmannosamine-6-phosphate 2-epimerase (NanE) has been proposed to use a deprotonation–reprotonation mechanism, with an essential catalytic lysine required for both steps. However, the structural determinants of this mechanism are not clearly established. We characterized NanE from Staphylococcus aureus using a new coupled assay to monitor NanE catalysis in real time and found that it has kinetic constants comparable with other species.Catalytic mechanism of ancestral L-lysine oxidase assigned by sequence data mining
A large number of protein sequences are registered in public databases such as PubMed. Functionally uncharacterized enzymes are included in these databases, some of which likely have potential for industrial applications. However, assignment of the enzymes remained difficult tasks for now. In this study, we assigned a total of 28 original sequences to uncharacterized enzymes in the FAD-dependent oxidase family expressed in some species of bacteria including Chryseobacterium, Flavobacterium, and Pedobactor.Structural insights into a flavin-dependent dehalogenase HadA explain catalysis and substrate inhibition via quadruple π-stacking
HadA is a flavin-dependent monooxygenase catalyzing hydroxylation plus dehalogenation/denitration, which is useful for biodetoxification and biodetection. In this study, the X-ray structure of wild-type HadA (HadAWT) co-complexed with reduced FAD (FADH–) and 4-nitrophenol (4NP) (HadAWT−FADH–−4NP) was solved at 2.3-Å resolution, providing the first full package (with flavin and substrate bound) structure of a monooxygenase of this type. Residues Arg101, Gln158, Arg161, Thr193, Asp254, Arg233, and Arg439 constitute a flavin-binding pocket, whereas the 4NP-binding pocket contains the aromatic side chain of Phe206, which provides π-π stacking and also is a part of the hydrophobic pocket formed by Phe155, Phe286, Thr449, and Leu457.Mammalian-like type II glutaminyl cyclases in Porphyromonas gingivalis and other oral pathogenic bacteria as targets for treatment of periodontitis
The development of a targeted therapy would significantly improve the treatment of periodontitis and its associated diseases including Alzheimer’s disease, rheumatoid arthritis, and cardiovascular diseases. Glutaminyl cyclases (QCs) from the oral pathogens Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia represent attractive target enzymes for small-molecule inhibitor development, as their action is likely to stabilize essential periplasmic and outer membrane proteins by N-terminal pyroglutamination.Modifying the resolving cysteine affects the structure and hydrogen peroxide reactivity of peroxiredoxin 2
Peroxiredoxin 2 (Prdx2) is a thiol peroxidase with an active site Cys (C52) that reacts rapidly with H2O2 and other peroxides. The sulfenic acid product condenses with the resolving Cys (C172) to form a disulfide which is recycled by thioredoxin or GSH via mixed disulfide intermediates or undergoes hyperoxidation to the sulfinic acid. C172 lies near the C terminus, outside the active site. It is not established whether structural changes in this region, such as mixed disulfide formation, affect H2O2 reactivity.A polysaccharide utilization locus from the gut bacterium Dysgonomonas mossii encodes functionally distinct carbohydrate esterases
The gut microbiota plays a central role in human health by enzymatically degrading dietary fiber and concomitantly excreting short chain fatty acids that are associated with manifold health benefits. The polysaccharide xylan is abundant in dietary fiber but noncarbohydrate decorations hinder efficient cleavage by glycoside hydrolases (GHs) and need to be addressed by carbohydrate esterases (CEs). Enzymes from carbohydrate esterase families 1 and 6 (CE1 and 6) perform key roles in xylan degradation by removing feruloyl and acetate decorations, yet little is known about these enzyme families especially with regard to their diversity in activity.A versatile cis-prenyltransferase from Methanosarcina mazei catalyzes both C- and O-prenylations
Polyprenyl groups, products of isoprenoid metabolism, are utilized in peptidoglycan biosynthesis, protein N-glycosylation, and other processes. These groups are formed by cis-prenyltransferases, which use allylic prenyl pyrophosphates as prenyl-donors to catalyze the C-prenylation of the general acceptor substrate, isopentenyl pyrophosphate. Repetition of this reaction forms (Z,E-mixed)-polyprenyl pyrophosphates, which are converted later into glycosyl carrier lipids, such as undecaprenyl phosphate and dolichyl phosphate.Identification and characterization of the pyridoxal 5’-phosphate allosteric site in Escherichia coli pyridoxine 5’-phosphate oxidase
Pyridoxal 5’-phosphate (PLP), the catalytically active form of vitamin B6, plays a pivotal role in metabolism as an enzyme cofactor. PLP is a very reactive molecule and can be very toxic unless its intracellular concentration is finely regulated. In Escherichia coli, PLP formation is catalyzed by pyridoxine 5’-phosphate oxidase (PNPO), a homodimeric FMN-dependent enzyme that is responsible for the last step of PLP biosynthesis and is also involved in the PLP salvage pathway. We have recently observed that E. coli PNPO undergoes an allosteric feedback inhibition by PLP, caused by a strong allosteric coupling between PLP binding at the allosteric site and substrate binding at the active site.Ancestral reconstruction of mammalian FMO1 enables structural determination, revealing unique features that explain its catalytic properties
Mammals rely on the oxidative flavin-containing monooxygenases (FMOs) to detoxify numerous and potentially deleterious xenobiotics; this activity extends to many drugs, giving FMOs high pharmacological relevance. However, our knowledge regarding these membrane-bound enzymes has been greatly impeded by the lack of structural information. We anticipated that ancestral-sequence reconstruction could help us identify protein sequences that are more amenable to structural analysis. As such, we hereby reconstructed the mammalian ancestral protein sequences of both FMO1 and FMO4, denoted as ancestral flavin-containing monooxygenase (AncFMO)1 and AncFMO4, respectively.Evolving the naturally compromised chorismate mutase from Mycobacterium tuberculosis to top performance
Chorismate mutase (CM), an essential enzyme at the branch-point of the shikimate pathway, is required for the biosynthesis of phenylalanine and tyrosine in bacteria, archaea, plants, and fungi. MtCM, the CM from Mycobacterium tuberculosis, has less than 1% of the catalytic efficiency of a typical natural CM and requires complex formation with 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase for high activity. To explore the full potential of MtCM for catalyzing its native reaction, we applied diverse iterative cycles of mutagenesis and selection, thereby raising kcat/Km 270-fold to 5 × 105m−1s−1, which is even higher than for the complex.
