ABSTRACT
SignificanceMore than 400 million tons of plastic waste is produced each year, the overwhelming majority of which ends up in landfills. Bioconversion strategies aimed at plastics have emerged as important components of enabling a circular economy for synthetic plastics, especially those that exhibit chemically similar linkages to those found in nature, such as polyesters. The enzyme system described in this work is essential for mineralization of the xenobiotic components of poly(ethylene terephthalate) (PET) in the biosphere. Our description of its structure and substrate preferences lays the groundwork for in vivo or ex vivo engineering of this system for PET upcycling.
Subject(s)
Dioxygenases , Phthalic Acids , Plastics/chemistry , Polyethylene Terephthalates/chemistryABSTRACT
Mercaptoethane sulfonate or coenzyme M (CoM) is the smallest known organic cofactor and is most commonly associated with the methane-forming step in all methanogenic archaea but is also associated with the anaerobic oxidation of methane to CO2 in anaerobic methanotrophic archaea and the oxidation of short-chain alkanes in Syntrophoarchaeum species. It has also been found in a small number of bacteria capable of the metabolism of small organics. Although many of the steps for CoM biosynthesis in methanogenic archaea have been elucidated, a complete pathway for the biosynthesis of CoM in archaea or bacteria has not been reported. Here, we present the complete CoM biosynthesis pathway in bacteria, revealing distinct chemical steps relative to CoM biosynthesis in methanogenic archaea. The existence of different pathways represents a profound instance of convergent evolution. The five-step pathway involves the addition of sulfite, the elimination of phosphate, decarboxylation, thiolation, and the reduction to affect the sequential conversion of phosphoenolpyruvate to CoM. The salient features of the pathway demonstrate reactivities for members of large aspartase/fumarase and pyridoxal 5'-phosphate-dependent enzyme families.
Subject(s)
Bacteria , Coenzymes , Euryarchaeota , Mesna , Anaerobiosis , Archaea/metabolism , Bacteria/metabolism , Coenzymes/biosynthesis , Euryarchaeota/metabolism , Mesna/metabolism , Methane/metabolism , Oxidation-Reduction , Phosphates/metabolismABSTRACT
2-Ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) is a member of the flavin and cysteine disulfide containing oxidoreductase family (DSOR) that catalyzes the unique reaction between atmospheric CO2 and a ketone/enolate nucleophile to generate acetoacetate. However, the mechanism of this reaction is not well understood. Here, we present evidence that 2-KPCC, in contrast to the well-characterized DSOR enzyme glutathione reductase, undergoes conformational changes during catalysis. Using a suite of biophysical techniques including limited proteolysis, differential scanning fluorimetry, and native mass spectrometry in the presence of substrates and inhibitors, we observed conformational differences between different ligand-bound 2-KPCC species within the catalytic cycle. Analysis of site-specific amino acid variants indicated that 2-KPCC-defining residues, Phe501-His506, within the active site are important for transducing these ligand induced conformational changes. We propose that these conformational changes promote substrate discrimination between H+ and CO2 to favor the metabolically preferred carboxylation product, acetoacetate.
Subject(s)
Carboxy-Lyases , Mesna , Acetoacetates/metabolism , Carbon Dioxide/metabolism , Carboxy-Lyases/metabolism , Catalysis , Ligands , Mesna/metabolism , Oxidoreductases/metabolism , Xanthobacter/metabolismABSTRACT
Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of monocopper enzymes broadly distributed across the tree of life. Recent reports indicate that LPMOs can use H2O2 as an oxidant and thus carry out a novel type of peroxygenase reaction involving unprecedented copper chemistry. Here, we present a combined computational and experimental analysis of the H2O2-mediated reaction mechanism. In silico studies, based on a model of the enzyme in complex with a crystalline substrate, suggest that a network of hydrogen bonds, involving both the enzyme and the substrate, brings H2O2 into a strained reactive conformation and guides a derived hydroxyl radical toward formation of a copper-oxyl intermediate. The initial cleavage of H2O2 and subsequent hydrogen atom abstraction from chitin by the copper-oxyl intermediate are the main energy barriers. Stopped-flow fluorimetry experiments demonstrated that the priming reduction of LPMO-Cu(II) to LPMO-Cu(I) is a fast process compared to the reoxidation reactions. Using conditions resulting in single oxidative events, we found that reoxidation of LPMO-Cu(I) is 2,000-fold faster with H2O2 than with O2, the latter being several orders of magnitude slower than rates reported for other monooxygenases. The presence of substrate accelerated reoxidation by H2O2, whereas reoxidation by O2 became slower, supporting the peroxygenase paradigm. These insights into the peroxygenase nature of LPMOs will aid in the development and application of enzymatic and synthetic copper catalysts and contribute to a further understanding of the roles of LPMOs in nature, varying from biomass conversion to chitinolytic pathogenesis-defense mechanisms.
Subject(s)
Bacterial Proteins/metabolism , Chitin/metabolism , Mixed Function Oxygenases/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Biocatalysis , Cellulose/chemistry , Cellulose/metabolism , Chitin/chemistry , Copper/chemistry , Copper/metabolism , Hydrogen Peroxide/chemistry , Hydrogen Peroxide/metabolism , Mixed Function Oxygenases/chemistry , Mixed Function Oxygenases/genetics , Oxidation-Reduction , Serratia marcescens/enzymologyABSTRACT
The 2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) enzyme is the only member of the disulfide oxidoreductase (DSOR) family of enzymes, which are important for reductively cleaving S-S bonds, to have carboxylation activity. 2-KPCC catalyzes the conversion of 2-ketopropyl-coenzyme M to acetoacetate, which is used as a carbon source, in a controlled reaction to exclude protons. A conserved His-Glu motif present in DSORs is key in the protonation step; however, in 2-KPCC, the dyad is substituted by Phe-His. Here, we propose that this difference is important for coupling carboxylation with C-S bond cleavage. We substituted the Phe-His dyad in 2-KPCC to be more DSOR like, replacing the phenylalanine with histidine (F501H) and the histidine with glutamate (H506E), and solved crystal structures of F501H and the double variant F501H_H506E. We found that F501 protects the enolacetone intermediate from protons and that the F501H variant strongly promotes protonation. We also provided evidence for the involvement of the H506 residue in stabilizing the developing charge during the formation of acetoacetate, which acts as a product inhibitor in the WT but not the H506E variant enzymes. Finally, we determined that the F501H substitution promotes a DSOR-like charge transfer interaction with flavin adenine dinucleotide, eliminating the need for cysteine as an internal base. Taken together, these results indicate that the 2-KPCC dyad is responsible for selectively promoting carboxylation and inhibiting protonation in the formation of acetoacetate.
Subject(s)
Dipeptides , Ketone Oxidoreductases , Mesna , Carboxy-Lyases/metabolism , Catalytic Domain , Oxidoreductases/metabolism , Substrate Specificity , Xanthobacter/metabolismABSTRACT
Microbial conversion of aromatic compounds is an emerging and promising strategy for valorization of the plant biopolymer lignin. A critical and often rate-limiting reaction in aromatic catabolism is O-aryl-demethylation of the abundant aromatic methoxy groups in lignin to form diols, which enables subsequent oxidative aromatic ring-opening. Recently, a cytochrome P450 system, GcoAB, was discovered to demethylate guaiacol (2-methoxyphenol), which can be produced from coniferyl alcohol-derived lignin, to form catechol. However, native GcoAB has minimal ability to demethylate syringol (2,6-dimethoxyphenol), the analogous compound that can be produced from sinapyl alcohol-derived lignin. Despite the abundance of sinapyl alcohol-based lignin in plants, no pathway for syringol catabolism has been reported to date. Here we used structure-guided protein engineering to enable microbial syringol utilization with GcoAB. Specifically, a phenylalanine residue (GcoA-F169) interferes with the binding of syringol in the active site, and on mutation to smaller amino acids, efficient syringol O-demethylation is achieved. Crystallography indicates that syringol adopts a productive binding pose in the variant, which molecular dynamics simulations trace to the elimination of steric clash between the highly flexible side chain of GcoA-F169 and the additional methoxy group of syringol. Finally, we demonstrate in vivo syringol turnover in Pseudomonas putida KT2440 with the GcoA-F169A variant. Taken together, our findings highlight the significant potential and plasticity of cytochrome P450 aromatic O-demethylases in the biological conversion of lignin-derived aromatic compounds.
Subject(s)
Cytochrome P-450 Enzyme System/genetics , Lignin/genetics , Protein Engineering , Pyrogallol/analogs & derivatives , Cytochrome P-450 Enzyme System/chemistry , Lignin/biosynthesis , Lignin/metabolism , Methylation , Oxidation-Reduction , Oxidoreductases, O-Demethylating/chemistry , Oxidoreductases, O-Demethylating/genetics , Pseudomonas putida/enzymology , Pseudomonas putida/genetics , Pyrogallol/chemistry , Pyrogallol/metabolismABSTRACT
Archaeal methanogens, methanotrophs, and alkanotrophs have a high demand for iron (Fe) and sulfur (S); however, little is known of how they acquire, traffic, deploy, and store these elements. Here, we examined the distribution of homologs of proteins mediating key steps in Fe/S metabolism in model microorganisms, including iron(II) sensing/uptake (FeoAB), sulfide extraction from cysteine (SufS), and the biosynthesis of iron-sulfur [Fe-S] clusters (SufBCDE), siroheme (Pch2 dehydrogenase), protoheme (AhbABCD), cytochrome c (Cyt c) (CcmCF), and iron storage/detoxification (Bfr, FtrA, and IssA), among 326 publicly available, complete or metagenome-assembled genomes of archaeal methanogens/methanotrophs/alkanotrophs. The results indicate several prevalent but nonuniversal features, including FeoB, SufBC, and the biosynthetic apparatus for the basic tetrapyrrole scaffold, as well as its siroheme (and F430) derivatives. However, several early-diverging genomes lacked SufS and pathways to synthesize and deploy heme. Genomes encoding complete versus incomplete heme biosynthetic pathways exhibited equivalent prevalences of [Fe-S] cluster binding proteins, suggesting an expansion of catalytic capabilities rather than substitution of heme for [Fe-S] in the former group. Several strains with heme binding proteins lacked heme biosynthesis capabilities, while other strains with siroheme biosynthesis capability lacked homologs of known siroheme binding proteins, indicating heme auxotrophy and unknown siroheme biochemistry, respectively. While ferritin proteins involved in ferric oxide storage were widespread, those involved in storing Fe as thioferrate were unevenly distributed. Collectively, the results suggest that differences in the mechanisms of Fe and S acquisition, deployment, and storage have accompanied the diversification of methanogens/methanotrophs/alkanotrophs, possibly in response to differential availability of these elements as these organisms evolved. IMPORTANCE Archaeal methanogens, methanotrophs, and alkanotrophs, argued to be among the most ancient forms of life, have a high demand for iron (Fe) and sulfur (S) for cofactor biosynthesis, among other uses. Here, using comparative bioinformatic approaches applied to 326 genomes, we show that major differences in Fe/S acquisition, trafficking, deployment, and storage exist in this group. Variation in these characters was generally congruent with the phylogenetic placement of these genomes, indicating that variation in Fe/S usage and deployment has contributed to the diversification and ecology of these organisms. However, incongruency was observed among the distribution of cofactor biosynthesis pathways and known protein destinations for those cofactors, suggesting auxotrophy or yet-to-be-discovered pathways for cofactor biosynthesis.
Subject(s)
Alkanes/metabolism , Archaea/classification , Archaea/metabolism , Coenzymes/metabolism , Iron/metabolism , Methane/metabolism , Sulfur/metabolism , Archaea/genetics , Archaea/isolation & purification , Archaeal Proteins/genetics , Archaeal Proteins/metabolism , Autotrophic Processes , Biosynthetic Pathways , Cysteine/metabolism , Ferric Compounds/metabolism , Heme/analogs & derivatives , Heme/metabolism , Iron-Sulfur Proteins/metabolism , PhylogenyABSTRACT
Iron is an essential micronutrient for life. In mammals, dietary iron is absorbed primarily in the small intestine. Currently, the impacts of dietary iron on the taxonomic structure and function of the gut microbiome and reciprocal effects on the animal host are not well understood. Here, we establish a mouse model of low-iron challenge in which intestinal biomarkers and reduced fecal iron reveal iron stress while serum iron and mouse behavioral markers indicate maintenance of iron homeostasis. We show that the diversity of the gut microbiome in conventional C57BL/6 mice changes dramatically during 2 weeks on a low-iron diet. We also show the effects of a low-iron diet on microbiome diversity are long lasting and not easily recovered when iron is returned to the diet. Finally, after optimizing taxon association methods, we show that some bacteria are unable to fully recover after the low-iron challenge and appear to be extirpated from the gut entirely. In particular, operational taxonomic units (OTUs) from the Prevotellaceae and Porphyromonadaceae families and Bacteroidales order are highly sensitive to low-iron conditions, while other seemingly insensitive OTUs recover. These results provide new insights into the iron requirements of gut microbiome members and add to the growing understanding of mammalian iron cycling.IMPORTANCE All cells need iron. Both too much and too little iron lead to diseases and unwanted outcomes. Although the impact of dietary iron on human cells and tissues has been well studied, there is currently a lack of understanding about how different levels of iron influence the abundant and diverse members of the human microbiome. This study develops a well-characterized mouse model for studying low-iron levels and identifies key groups of bacteria that are most affected. We found that the microbiome undergoes large changes when iron is removed from the diet but that many individual bacteria are able to rebound when iron levels are changed back to normal. That said, a select few members, referred to as iron-sensitive bacteria, seem to be lost. This study begins to identify individual members of the mammalian microbiome most affected by changes in dietary iron levels.
Subject(s)
Gastrointestinal Microbiome/drug effects , Iron/administration & dosage , Animals , Bacteria/drug effects , Bacteria/genetics , Bacteria/isolation & purification , Feces/chemistry , Female , Gastrointestinal Microbiome/genetics , Iron/blood , Iron/pharmacokinetics , Male , Mice, Inbred C57BL , RNA, Ribosomal, 16SABSTRACT
Alkenes and ketones are two classes of ubiquitous, toxic organic compounds in natural environments produced in several biological and anthropogenic processes. In spite of their toxicity, these compounds are utilized as primary carbon and energy sources or are generated as intermediate metabolites in the metabolism of other compounds by many diverse bacteria. The aerobic metabolism of some of the smallest and most volatile of these compounds (propylene, acetone, isopropanol) involves novel carboxylation reactions resulting in a common product acetoacetate. Propylene is metabolized in a four-step pathway involving five enzymes where the penultimate step is a carboxylation reaction catalyzed by a unique disulfide oxidoreductase that couples reductive cleavage of a thioether linkage with carboxylation to produce acetoacetate. The carboxylation of isopropanol begins with conversion to acetone via an alcohol dehydrogenase. Acetone is converted to acetoacetate in a single step by an acetone carboxylase which couples the hydrolysis of MgATP to the activation of both acetone and bicarbonate, generating highly reactive intermediates that are condensed into acetoacetate at a Mn2+ containing the active site. Acetoacetate is then utilized in central metabolism where it is readily converted to acetyl-coenzyme A and subsequently converted into biomass or utilized in energy metabolism via the tricarboxylic acid cycle. This review summarizes recent structural and biochemical findings that have contributed significant insights into the mechanism of these two unique carboxylating enzymes.
Subject(s)
Acetone/metabolism , Alkenes/metabolism , Bacteria/metabolism , 2-Propanol/metabolism , Acetoacetates/metabolism , Acetyl Coenzyme A/metabolism , Bicarbonates/metabolism , Catalysis , Citric Acid Cycle/physiologyABSTRACT
NADPH: 2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) is a bacterial disulfide oxidoreductase (DSOR) that, uniquely in this family, catalyzes CO2 fixation. 2-KPCC differs from other DSORs by having a phenylalanine that replaces a conserved histidine, which in typical DSORs is essential for stabilizing the reduced, reactive form of the active site. Here, using site-directed mutagenesis and stopped-flow kinetics, we examined the reactive form of 2-KPCC and its single turnover reactions with a suicide substrate and CO2 The reductive half-reaction of 2-KPCC was kinetically and spectroscopically similar to that of a typical DSOR, GSH reductase, in which the active-site histidine had been replaced with an alanine. However, the reduced, reactive form of 2-KPCC was distinct from those typical DSORs. In the absence of the histidine, the flavin and disulfide moieties were no longer coupled via a covalent or charge transfer interaction as in typical DSORs. Similar to thioredoxins, the pKa between 7.5 and 8.1 that controls reactivity appeared to be due to a single proton shared between the cysteines of the dithiol, which effectively stabilizes the attacking cysteine sulfide and renders it capable of breaking the strong C-S bond of the substrate. The lack of a histidine protected 2-KPCC's reactive intermediate from unwanted protonation; however, without its input as a catalytic acid-base, the oxidative half-reaction where carboxylation takes place was remarkably slow, limiting the overall reaction rate. We conclude that stringent regulation of protons in the DSOR active site supports C-S bond cleavage and selectivity for CO2 fixation.
Subject(s)
Carbon Dioxide/metabolism , Ketone Oxidoreductases/metabolism , Xanthobacter/enzymology , Catalytic Domain , Ketone Oxidoreductases/chemistry , Kinetics , Models, Molecular , NADP/metabolism , Oxidation-Reduction , Substrate Specificity , Xanthobacter/chemistry , Xanthobacter/metabolismABSTRACT
Molecular oxygen (O2)-utilizing enzymes are among the most important in biology. The abundance of O2, its thermodynamic power, and the benign nature of its end products have raised interest in oxidases and oxygenases for biotechnological applications. Although most O2-dependent enzymes have an absolute requirement for an O2-activating cofactor, several classes of oxidases and oxygenases accelerate direct reactions between substrate and O2 using only the protein environment. Nogalamycin monooxygenase (NMO) from Streptomyces nogalater is a cofactor-independent enzyme that catalyzes rate-limiting electron transfer between its substrate and O2 Here, using enzyme-kinetic, cyclic voltammetry, and mutagenesis methods, we demonstrate that NMO initially activates the substrate, lowering its pKa by 1.0 unit (ΔG* = 1.4 kcal mol-1). We found that the one-electron reduction potential, measured for the deprotonated substrate both inside and outside the protein environment, increases by 85 mV inside NMO, corresponding to a ΔΔG0' of 2.0 kcal mol-1 (0.087 eV) and that the activation barrier, ΔG, is lowered by 4.8 kcal mol-1 (0.21 eV). Applying the Marcus model, we observed that this suggests a sizable decrease of 28 kcal mol-1 (1.4 eV) in the reorganization energy (λ), which constitutes the major portion of the protein environment's effect in lowering the reaction barrier. A similar role for the protein has been proposed in several cofactor-dependent systems and may reflect a broader trend in O2-utilizing proteins. In summary, NMO's protein environment facilitates direct electron transfer, and NMO accelerates rate-limiting electron transfer by strongly lowering the reorganization energy.
Subject(s)
Mixed Function Oxygenases/metabolism , Nogalamycin/metabolism , Oxygen/metabolism , Catalytic Domain , Electron Transport , Mixed Function Oxygenases/chemistry , Mixed Function Oxygenases/genetics , Models, Molecular , Mutagenesis , Streptomyces/enzymology , Temperature , ThermodynamicsABSTRACT
Clostridium difficile is a Gram-positive, spore-forming anaerobic bacterium that infects the colon, causing symptoms ranging from infectious diarrhea to fulminant colitis. In the last decade, the number of C. difficile infections has dramatically risen, making it the leading cause of reported hospital acquired infection in the United States. Bacterial toxins produced during C. difficile infection (CDI) damage host epithelial cells, releasing erythrocytes and heme into the gastrointestinal lumen. The reactive nature of heme can lead to toxicity through membrane disruption, membrane protein and lipid oxidation, and DNA damage. Here we demonstrate that C. difficile detoxifies excess heme to achieve full virulence within the gastrointestinal lumen during infection, and that this detoxification occurs through the heme-responsive expression of the heme activated transporter system (HatRT). Heme-dependent transcriptional activation of hatRT was discovered through an RNA-sequencing analysis of C. difficile grown in the presence of a sub-toxic concentration of heme. HatRT is comprised of a TetR family transcriptional regulator (hatR) and a major facilitator superfamily transporter (hatT). Strains inactivated for hatR or hatT are more sensitive to heme toxicity than wild-type. HatR binds heme, which relieves the repression of the hatRT operon, whereas HatT functions as a heme efflux pump. In a murine model of CDI, a strain inactivated for hatT displayed lower pathogenicity in a toxin-independent manner. Taken together, these data suggest that HatR senses intracellular heme concentrations leading to increased expression of the hatRT operon and subsequent heme efflux by HatT during infection. These results describe a mechanism employed by C. difficile to relieve heme toxicity within the host, and set the stage for the development of therapeutic interventions to target this bacterial-specific system.
Subject(s)
Clostridioides difficile/genetics , Clostridioides difficile/pathogenicity , Clostridium Infections/microbiology , Heme/metabolism , Virulence/physiology , Animals , Bacterial Proteins/metabolism , Clostridioides difficile/metabolism , Clostridium Infections/metabolism , Genes, Bacterial/genetics , Male , Mice , Mice, Inbred C57BL , Operon/geneticsABSTRACT
The role of the chemical environment in promoting anthralin/O2 reactions was discovered using neat solvents to model the amino acids of a cofactor-independent oxygenase. Experimental and computational results highlight the importance of the substrate-enolate, which is accessed via energetically small, escalating steps in which the ground-state keto-isomer is tautomerized to an enol and then ionized by solvent. The resulting ion-pair is poised for spontaneous electron transfer to O2. Similar activation may be exploited in biological/nonbiological oxidations involving O2.
ABSTRACT
The pathway for assembling heme ends with a unique set of enzymes in Gram-positive bacteria. Substrates for these reactions include coproporphyrin III, Fe(II), and H2O2, which are highly reactive and toxic. Because these bacteria lack membranous compartments, we hypothesized that metabolite flux may occur via a transient protein-protein interaction between the final two pathway enzymes, coproporphyrin ferrochelatase (CpfC) and coproheme decarboxylase (ChdC). This hypothesis was tested using enzymes from the pathogen Staphylococcus aureus and a corresponding ΔchdC knockout strain. The ultraviolet-visible spectral features of coproporphyrin III served as an in vitro indicator of a protein-protein interaction. A CpfC-ChdC KD of 17 ± 7 µM was determined, consistent with transient complexation and supported by the observation that the catalytic competence of both enzymes was moderately suppressed in the stable complex. The ΔchdC S. aureus was transformed with plasmids containing single-amino acid mutants in the active site gate of ChdC. The porphyrin content and growth phenotypes of these mutants showed that K129 and Y133 promote the ChdC-CpfC interaction and revealed the importance of E120. Understanding the nature of interactions between these enzymes and those further upstream in the heme biosynthesis pathway could provide new means of specifically targeting pathogenic Gram-positive bacteria such as S. aureus.
Subject(s)
Heme/biosynthesis , Staphylococcus aureus/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Coproporphyrins/metabolism , Phenotype , Point MutationABSTRACT
The H2O2-dependent oxidative decarboxylation of coproheme III is the final step in the biosynthesis of heme b in many microbes. However, the coproheme decarboxylase reaction mechanism is unclear. The structure of the decarboxylase in complex with coproheme III suggested that the substrate iron, reactive propionates, and an active-site tyrosine convey a net 2e-/2H+ from each propionate to an activated form of H2O2 Time-resolved EPR spectroscopy revealed that Tyr-145 formed a radical species within 30 s of the reaction of the enzyme-coproheme complex with H2O2 This radical disappeared over the next 270 s, consistent with a catalytic intermediate. Use of the harderoheme III intermediate as substrate or substitutions of redox-active side chains (W198F, W157F, or Y113S) did not strongly affect the appearance or intensity of the radical spectrum measured 30 s after initiating the reaction with H2O2, nor did it change the â¼270 s required for the radical signal to recede to ≤10% of its initial intensity. These results suggested Tyr-145 as the site of a catalytic radical involved in decarboxylating both propionates. Tyr-145⢠was accompanied by partial loss of the initially present Fe(III) EPR signal intensity, consistent with the possible formation of Fe(IV)=O. Site-specifically deuterated coproheme gave rise to a kinetic isotope effect of â¼2 on the decarboxylation rate constant, indicating that cleavage of the propionate Cß-H bond was partly rate-limiting. The inferred mechanism requires two consecutive hydrogen atom transfers, first from Tyr-145 to the substrate Fe/H2O2 intermediate and then from the propionate Cß-H to Tyr-145â¢.
Subject(s)
Carboxy-Lyases/metabolism , Ferric Compounds/chemistry , Free Radicals/chemistry , Heme/metabolism , Hydrogen Peroxide/chemistry , Propionates/chemistry , Tyrosine/chemistry , Carboxy-Lyases/genetics , Catalysis , Catalytic Domain , Crystallography, X-Ray , Decarboxylation , Electron Spin Resonance Spectroscopy , Heme/chemistry , Kinetics , Models, Molecular , Mutation , Oxidation-ReductionABSTRACT
For nearly 30 years, coenzyme M (CoM) was assumed to be present solely in methanogenic archaea. In the late 1990s, CoM was reported to play a role in bacterial propene metabolism, but no biosynthetic pathway for CoM has yet been identified in bacteria. Here, using bioinformatics and proteomic approaches in the metabolically versatile bacterium Xanthobacter autotrophicus Py2, we identified four putative CoM biosynthetic enzymes encoded by the xcbB1, C1, D1, and E1 genes. Only XcbB1 was homologous to a known CoM biosynthetic enzyme (ComA), indicating that CoM biosynthesis in bacteria involves enzymes different from those in archaea. We verified that the ComA homolog produces phosphosulfolactate from phosphoenolpyruvate (PEP), demonstrating that bacterial CoM biosynthesis is initiated similarly as the phosphoenolpyruvate-dependent methanogenic archaeal pathway. The bioinformatics analysis revealed that XcbC1 and D1 are members of the aspartase/fumarase superfamily (AFS) and that XcbE1 is a pyridoxal 5'-phosphate-containing enzyme with homology to d-cysteine desulfhydrases. Known AFS members catalyze ß-elimination reactions of succinyl-containing substrates, yielding fumarate as the common unsaturated elimination product. Unexpectedly, we found that XcbC1 catalyzes ß-elimination on phosphosulfolactate, yielding inorganic phosphate and a novel metabolite, sulfoacrylic acid. Phosphate-releasing ß-elimination reactions are unprecedented among the AFS, indicating that XcbC1 is an unusual phosphatase. Direct demonstration of phosphosulfolactate synthase activity for XcbB1 and phosphate ß-elimination activity for XcbC1 strengthened their hypothetical assignment to a CoM biosynthetic pathway and suggested functions also for XcbD1 and E1. Our results represent a critical first step toward elucidating the CoM pathway in bacteria.
Subject(s)
Mesna/metabolism , Phosphates/metabolism , Xanthobacter/metabolism , Aspartate Ammonia-Lyase/metabolism , Bacteria/metabolism , Computational Biology/methods , Crystallography, X-Ray , Fumarate Hydratase/metabolism , Fumarates , Phosphoenolpyruvate/metabolism , Phosphoric Acids , Phosphoric Monoester Hydrolases , Proteomics , Pyridoxal PhosphateABSTRACT
O2-evolving chlorite dismutases (Clds) efficiently convert chlorite (ClO2-) to O2 and Cl-. Dechloromonas aromatica Cld ( DaCld) is a highly active chlorite-decomposing homopentameric enzyme, typical of Clds found in perchlorate- and chlorate-respiring bacteria. The Gram-negative, human pathogen Klebsiella pneumoniae contains a homodimeric Cld ( KpCld) that also decomposes ClO2-, albeit with an activity 10-fold lower and a turnover number lower than those of DaCld. The interactions between the distal pocket and heme ligand of the DaCld and KpCld active sites have been probed via kinetic, thermodynamic, and spectroscopic behaviors of their cyanide complexes for insight into active site characteristics that are deterministic for chlorite decomposition. At 4.7 × 10-9 M, the KD for the KpCld-CN- complex is 2 orders of magnitude smaller than that of DaCld-CN- and indicates an affinity for CN- that is greater than that of most heme proteins. The difference in CN- affinity between Kp- and DaClds is predominantly due to differences in koff. The kinetics of binding of cyanide to DaCld, DaCld(R183Q), and KpCld between pH 4 and 8.5 corroborate the importance of distal Arg183 and a p Ka of â¼7 in stabilizing complexes of anionic ligands, including the substrate. The Fe-C stretching and FeCN bending modes of the DaCld-CN- (νFe-C, 441 cm-1; δFeCN, 396 cm-1) and KpCld-CN- (νFe-C, 441 cm-1; δFeCN, 356 cm-1) complexes reveal differences in their FeCN angle, which suggest different distal pocket interactions with their bound cyanide. Conformational differences in their catalytic sites are also reported by the single ferrous KpCld carbonyl complex, which is in contrast to the two conformers observed for DaCld-CO.
Subject(s)
Cyanides/chemistry , Cyanides/metabolism , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Catalytic Domain , Chlorides/metabolism , Heme/chemistry , Heme/metabolism , Klebsiella pneumoniae/enzymology , Klebsiella pneumoniae/metabolism , Models, Molecular , Oxygen/metabolismABSTRACT
O2-evolving chlorite dismutases (Clds) fall into two subfamilies, which efficiently convert ClO2- to O2 and Cl-. The Cld from Dechloromonas aromatica (DaCld) represents the chlorite-decomposing homopentameric enzymes found in perchlorate- and chlorate-respiring bacteria. The Cld from the Gram-negative human pathogen Klebsiella pneumoniae (KpCld) is representative of the second subfamily, comprising homodimeric enzymes having truncated N-termini. Here steric and nonbonding properties of the DaCld and KpCld active sites have been probed via kinetic, thermodynamic, and spectroscopic behaviors of their fluorides, chlorides, and hydroxides. Cooperative binding of Cl- to KpCld drives formation of a hexacoordinate, high-spin aqua heme, whereas DaCld remains pentacoordinate and high-spin under analogous conditions. Fluoride coordinates to the heme iron in KpCld and DaCld, exhibiting ν(FeIII-F) bands at 385 and 390 cm-1, respectively. Correlation of these frequencies with their CT1 energies reveals strong H-bond donation to the F- ligand, indicating that atoms directly coordinated to heme iron are accessible to distal H-bond donation. New vibrational frequency correlations between either ν(FeIII-F) or ν(FeIII-OH) and ν(FeII-His) of Clds and other heme proteins are reported. These correlations orthogonalize proximal and distal effects on the bonding between iron and exogenous π-donor ligands. The axial Fe-X vibrations and the relationships between them illuminate both similarities and differences in the H-bonding and electrostatic properties of the distal and proximal heme environments in pentameric and dimeric Clds. Moreover, they provide general insight into the structural basis of reactivity toward substrates in heme-dependent enzymes and their mechanistic intermediates, especially those containing the ferryl moiety.
Subject(s)
Bacterial Proteins/chemistry , Chlorides/chemistry , Fluorides/chemistry , Klebsiella pneumoniae/enzymology , Oxidoreductases/chemistry , Peroxides/chemistry , Catalytic Domain , Heme/chemistry , Hydrogen Bonding , Oxygen/chemistryABSTRACT
A recently discovered pathway for the biosynthesis of heme b ends in an unusual reaction catalyzed by coproheme decarboxylase (HemQ), where the Fe(II)-containing coproheme acts as both substrate and cofactor. Because both O2 and H2O2 are available as cellular oxidants, pathways for the reaction involving either can be proposed. Analysis of reaction kinetics and products showed that, under aerobic conditions, the ferrous coproheme-decarboxylase complex is rapidly and selectively oxidized by O2 to the ferric state. The subsequent second-order reaction between the ferric complex and H2O2 is slow, pH-dependent, and further decelerated by D2O2 (average kinetic isotope effect of 2.2). The observation of rapid reactivity with peracetic acid suggested the possible involvement of Compound I (ferryl porphyrin cation radical), consistent with coproheme and harderoheme reduction potentials in the range of heme proteins that heterolytically cleave H2O2. Resonance Raman spectroscopy nonetheless indicated a remarkably weak Fe-His interaction; how the active site structure may support heterolytic H2O2 cleavage is therefore unclear. From a cellular perspective, the use of H2O2 as an oxidant in a catalase-positive organism is intriguing, as is the unusual generation of heme b in the Fe(III) rather than Fe(II) state as the end product of heme synthesis.
Subject(s)
Bacterial Proteins/metabolism , Carboxy-Lyases/metabolism , Heme/metabolism , Hemin/analogs & derivatives , Hydrogen Peroxide/metabolism , Oxygen/metabolism , Aerobiosis , Bacterial Proteins/chemistry , Biosynthetic Pathways , Carboxy-Lyases/chemistry , Catalase/metabolism , Ferric Compounds/chemistry , Ferric Compounds/metabolism , Ferrous Compounds/chemistry , Ferrous Compounds/metabolism , Heme/chemistry , Hemin/chemistry , Hemin/metabolism , Hydrogen-Ion Concentration , Kinetics , Models, Chemical , Molecular Structure , Oxidation-Reduction , Peracetic Acid/metabolism , Spectrophotometry , Spectrum Analysis, Raman , Staphylococcus aureus/enzymology , Staphylococcus aureus/metabolismABSTRACT
Members of the antibiotic biosynthesis monooxygenase family catalyze O2-dependent oxidations and oxygenations in the absence of any metallo- or organic cofactor. How these enzymes surmount the kinetic barrier to reactions between singlet substrates and triplet O2 is unclear, but the reactions have been proposed to occur via a flavin-like mechanism, where the substrate acts in lieu of a flavin cofactor. To test this model, we monitored the uncatalyzed and enzymatic reactions of dithranol, a substrate for the nogalamycin monooxygenase (NMO) from Streptomyces nogalater As with flavin, dithranol oxidation was faster at a higher pH, although the reaction did not appear to be base-catalyzed. Rather, conserved asparagines contributed to suppression of the substrate pKa The same residues were critical for enzymatic catalysis that, consistent with the flavoenzyme model, occurred via an O2-dependent slow step. Evidence for a superoxide/substrate radical pair intermediate came from detection of enzyme-bound superoxide during turnover. Small molecule and enzymatic superoxide traps suppressed formation of the oxygenation product under uncatalyzed conditions, whereas only the small molecule trap had an effect in the presence of NMO. This suggested that NMO both accelerated the formation and directed the recombination of a superoxide/dithranyl radical pair. These catalytic strategies are in some ways flavin-like and stand in contrast to the mechanisms of urate oxidase and (1H)-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, both cofactor-independent enzymes that surmount the barriers to direct substrate/O2 reactivity via markedly different means.