Cobalamin-dependent Enzymes Research Articles

Transcriptomic and proteomic changes associated with cobalamin-dependent propionate production by the rumen bacterium Xylanibacter ruminicola.

In ruminants, the rumen microbial community plays a critical role in nutrition through the fermentation of feed to provide vital energy substrates for the host animal. Propionate is a major rumen fermentation end-product and increasing its production is desirable given its importance in host glucose production and impact on greenhouse gas production. Vitamin B12 (cobalamin) can induce propionate production in the prominent rumen bacterium Xylanibacter ruminicola, but it is not fully understood how cobalamin regulates propionate pathway activity. Contrary to expectation, we found that cobalamin supplementation had little effect on propionate pathway expression at transcriptome and proteome levels, with minor upregulation of genes encoding the cobalamin-dependent enzyme of the pathway. These findings provide new insights into factors that regulate propionate production and suggest that cobalamin-dependent propionate production by X. ruminicola is controlled post-translationally.

Growth requirement for methionine in human melanoma-derived cell lines with different levels of MMACHC expression and methylation

Methionine dependence, the inability to grow in culture when methionine in the medium is replaced by its metabolic precursor homocysteine, occurs in many tumor cell lines. In most affected lines, the cause of methionine dependence is not known. An exception is the melanoma-derived cell line MeWo-LC1, in which hypermethylation of the MMACHC gene is associated with decreased MMACHC expression. Decreased expression results in decreased provision of the methylcobalamin cofactor required for activity of methionine synthase and thus decreased conversion of homocysteine to methionine. Analysis of data in the Cancer Cell Line Encyclopedia Archive demonstrated that MMACHC hypermethylation and decreased MMACHC expression occurred more frequently in melanoma cell lines when compared to other tumor cell lines. We further investigated methionine dependence and aspects of MMACHC function in a panel of six melanoma lines, including both melanoma lines with known methionine dependence status (MeWo, which is methionine independent, and A375, which is methionine dependent). We found that the previously unclassified melanoma lines HMCB, Colo829 and SH-4 were methionine dependent, while SK-Mel-28 was methionine independent. However, despite varying levels of MMACHC methylation and expression, none of the tested lines had decreased methylcobalamin and adenosylcobalamin synthesis as seen in MeWo-LC1, and the functions of both cobalamin-dependent enzymes methionine synthase and methylmalonyl-CoA mutase were intact. Thus, while melanoma lines were characterized by relatively high levels of MMACHC methylation and low expression, the defect in metabolism observed in MeWo-LC1 was unique, and decreased MMACHC expression was not a cause of methionine dependence in the other melanoma lines.

Draft Genome Sequence of Pedococcus sp. Strain 5OH_020, Isolated from California Grassland Soil.

The draft genome sequence of the soil bacterium Pedococcus sp. strain 5OH_020, isolated on a natural cobalamin analog, comprises 4.4 Mbp, with 4,108 protein-coding genes. Its genome encodes cobalamin-dependent enzymes, including methionine synthase and class II ribonucleotide reductase. Taxonomic analysis suggests that it is a novel species within the genus Pedococcus.

Anaerobic demethylation of guaiacyl-derived monolignols enabled by a designed artificial cobalamin methyltransferase fusion enzyme.

Lignin-derived aryl methyl ethers (e.g. coniferyl alcohol, ferulic acid) are expected to be a future carbon source for chemistry. The well-known P450 dependent biocatalytic O-demethylation of these aryl methyl ethers is prone to side product formation especially for the oxidation sensitive catechol products which get easily oxidized in the presence of O2. Alternatively, biocatalytic demethylation using cobalamin dependent enzymes may be used under anaerobic conditions, whereby two proteins, namely a methyltransferase and a carrier protein are required. To make this approach applicable for preparative transformations, fusion proteins were designed connecting the cobalamin-dependent methyltransferase (MT) with the corrinoid-binding protein (CP) from Desulfitobacterium hafniense by variable glycine linkers. From the proteins created, the fusion enzyme MT-L5-CP with the shortest linker performed best of all fusion enzymes investigated showing comparable and, in some aspects, even better performance than the separated proteins. The fusion enzymes provided several advantages like that the cobalamin cofactor loading step required originally for the CP could be skipped enabling a significantly simpler protocol. Consequently, the biocatalytic demethylation was performed using Schlenk conditions allowing the O-demethylation e.g. of the monolignol coniferyl alcohol on a 25 mL scale leading to 75% conversion. The fusion enzyme represents a promising starting point to be evolved for alternative demethylation reactions to diversify natural products and to valorize lignin.

Metagenomic insights into the microbe-mediated B and K2 vitamin biosynthesis in the gastrointestinal microbiome of ruminants

BackgroundB and K2 vitamins, essential nutrients in host metabolism, can be synthesized by the rumen microbiome in ruminants and subsequently absorbed by the host. However, the B and K2 vitamin biosynthesis by the whole gastrointestinal microbiome and their abundances in different dietary strategies are largely unknown. Here, we reanalyzed our previous large-scale metagenomic data on the gastrointestinal microbiome of seven ruminant species and recruited 17,425 nonredundant microbial genomes from published datasets to gain a comprehensive understanding of the microbe-mediated B and K2 vitamin biosynthesis in ruminants.ResultsWe identified 1,135,807 genes and 167 enzymes involved in B and K2 vitamin biosynthesis. Our results indicated that the total abundances of B and K2 vitamin biosynthesis were dominant in the stomach microbiome, while the biosynthesis of thiamine, niacin, and pyridoxine was more abundant in the large intestine. By examining 17,425 nonredundant genomes, we identified 2366 high-quality genomes that were predicted to de novo biosynthesize at least one vitamin. Genomic analysis suggested that only 2.7% of these genomes can synthesize five or more vitamins, and nearly half of genomes can synthesize only one vitamin. Moreover, we found that most genomes possessed cobalamin transporters or cobalamin-dependent enzymes to consume cobalamin directly, and only a few microbial genomes possessed a complete cobalamin biosynthesis pathway. Based on these genomic data, we examined the effect of the high-grain (HG) diet on the vitamin biosynthesis of the rumen microbiome of dairy cattle. We revealed that most vitamin biosynthesis was enhanced in the HG group, while only cobalamin synthesis was inhibited in the HG group, indicating that dietary fiber is vital for cobalamin biosynthesis.ConclusionsWe primarily provided a gene catalog and 2366 microbial genomes involved in B and K2 vitamin biosynthesis in ruminants. Our findings demonstrated the regional heterogeneity and dietary effect of vitamin biosynthetic potential in the ruminant gastrointestinal microbiome and interpreted the biosynthesis mechanisms of these microbes and their physiological adaptability. This study expands our understanding of microbe-mediated vitamin biosynthesis in ruminants and may provide novel targets for manipulation to improve the production of these essential vitamins.6cdWEjSDW5ZW7DH8TeMqdwVideo abstract.

Membrane transport of cobalamin.

A wide variety of organisms encode cobalamin-dependent enzymes catalyzing essential metabolic reactions, but the cofactor cobalamin (vitamin B12) is only synthesized by a subset of bacteria and archaea. The biosynthesis of cobalamin is complex and energetically costly, making cobalamin variants and precursors metabolically valuable. Auxotrophs for these molecules have evolved uptake mechanisms to compensate for the lack of a synthesis pathway. Bacterial transport of cobalamin involves the passage over one or two lipidic membranes in Gram-positive and -negative bacteria, respectively. In higher eukaryotes, a complex system of carriers, receptors and transporters facilitates the delivery of the essential molecule to the tissues. Biochemical and genetic approaches have identified different transporter families involved in cobalamin transport. The majority of the characterized cobalamin transporters are active transport systems that belong to the ATP-binding cassette (ABC) superfamily of transporters. In this chapter, we describe the different cobalamin transport systems characterized to date that are present in bacteria and humans, as well as yet-to-be-identified transporters.

Methionine synthase supports tumour tetrahydrofolate pools.

Mammalian cells require activated folates to generate nucleotides for growth and division. The most abundant circulating folate species is 5-methyl tetrahydrofolate (5-methyl-THF), which is used to synthesize methionine from homocysteine via the cobalamin-dependent enzyme methionine synthase (MTR). Cobalamin deficiency traps folates as 5-methyl-THF. Here, we show using isotope tracing that methionine synthase is only a minor source of methionine in cell culture, tissues, or xenografted tumors. Instead, methionine synthase is required for cells to avoid folate trapping and assimilate 5-methyl-THF into other folate species. Under conditions of physiological extracellular folates, genetic MTR knockout in tumor cells leads to folate trapping, purine synthesis stalling, nucleotide depletion, and impaired growth in cell culture and as xenografts. These defects are rescued by free folate but not one-carbon unit supplementation. Thus, MTR plays a crucial role in liberating tetrahydrofolate for use in one-carbon metabolism.

De Novo Cobalamin Biosynthesis, Transport, and Assimilation and Cobalamin-Mediated Regulation of Methionine Biosynthesis in Mycobacterium smegmatis.

Cobalamin is an essential cofactor in all domains of life, yet its biosynthesis is restricted to some bacteria and archaea. Mycobacterium smegmatis, an environmental saprophyte frequently used as surrogate for the obligate human pathogen M. tuberculosis, carries approximately 30 genes predicted to be involved in de novo cobalamin biosynthesis. M. smegmatis also encodes multiple cobalamin-dependent enzymes, including MetH, a methionine synthase that catalyzes the final reaction in methionine biosynthesis. In addition to metH, M. smegmatis possesses a cobalamin-independent methionine synthase, metE, suggesting that enzyme use-MetH versus MetE-is regulated by cobalamin availability. Consistent with this notion, we previously described a cobalamin-sensing riboswitch controlling metE expression in M. tuberculosis Here, we apply a targeted mass spectrometry-based approach to confirm de novo cobalamin biosynthesis in M. smegmatis during aerobic growth in vitro We also demonstrate that M. smegmatis can transport and assimilate exogenous cyanocobalamin (CNCbl; also known as vitamin B12) and its precursor, dicyanocobinamide ([CN]2Cbi). However, the uptake of CNCbl and (CN)2Cbi in this organism is restricted and seems dependent on the conditional essentiality of the cobalamin-dependent methionine synthase. Using gene and protein expression analyses combined with single-cell growth kinetics and live-cell time-lapse microscopy, we show that transcription and translation of metE are strongly attenuated by endogenous cobalamin. These results support the inference that metH essentiality in M. smegmatis results from riboswitch-mediated repression of MetE expression. Moreover, differences observed in cobalamin-dependent metabolism between M. smegmatis and M. tuberculosis provide some insight into the selective pressures which might have shaped mycobacterial metabolism for pathogenicity.IMPORTANCE Alterations in cobalamin-dependent metabolism have marked the evolution of Mycobacterium tuberculosis into a human pathogen. However, the role(s) of cobalamin in mycobacterial physiology remains poorly understood. Using the nonpathogenic saprophyte M. smegmatis, we investigated the production of cobalamin, transport and assimilation of cobalamin precursors, and the role of cobalamin in regulating methionine biosynthesis. We confirm constitutive de novo cobalamin biosynthesis in M. smegmatis, in contrast with M. tuberculosis, which appears to lack de novo cobalamin biosynthetic capacity. We also show that uptake of cyanocobalamin (vitamin B12) and its precursors is restricted in M. smegmatis, apparently depending on the cofactor requirements of the cobalamin-dependent methionine synthase. These observations establish M. smegmatis as an informative foil to elucidate key metabolic adaptations enabling mycobacterial pathogenicity.

Utilization of cobalamin is ubiquitous in early-branching fungal phyla.

Cobalamin is a cofactor present in essential metabolic pathways in animals and one of the water-soluble vitamins. It is a complex compound synthesized solely by prokaryotes. Cobalamin dependence is scattered across the tree of life. In particular, fungi and plants were deemed devoid of cobalamin. We demonstrate that cobalamin is utilized by all non-Dikarya fungi lineages. This observation is supported by the genomic presence of both B12-dependent enzymes and cobalamin modifying enzymes. Fungal cobalamin-dependent enzymes are highly similar to their animal homologs. Phylogenetic analyses support a scenario of vertical inheritance of the cobalamin usage with several losses. Cobalamin usage was probably lost in Mucorinae and at the base of Dikarya which groups most of the model organisms and which hindered B12-dependent metabolism discovery in fungi. Our results indicate that cobalamin dependence was a widely distributed trait at least in Opisthokonta, across diverse microbial eukaryotes and was likely present in the LECA.

A New Approach for Second-tier Analysis of Methylmalonic Acid in Dried Blood Spots using Liquid Chromatography Tandem Mass Spectrometry

Introduction: Inactivity or diminished activity of an enzyme Methylmalonyl-CoA mutase (a Cobalamin dependent) enzyme causes inborn error of metabolism named Methylmalonic Acidemia/Aciduria (MMA). Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) based method for the diagnosis of MMA in Newborn Screening (NBS), is often challenging due to the nonspecificity of propionylcarnitine (C3), a primary marker in routine NBS. Aim: To develop a Flow Injection Analysis (FIA) method for the second-tier estimation of Methylmalonic acid in the Dried Blood Spots (DBS) of primary NBS. Materials and Methods: A retrospective NBS study was conducted for a period of two years i.e. (November 2015 to November 2017) at the Paediatrics Research and Genetic Lab of Maulana Azad Medical College associated Lok Nayak Hospital, a Tertiary Care Centre in New Delhi, India. DBS samples were collected by heel- prick method and a second tier detection, quantification of methylmalonic acid was performed by LC-MS/MS on all samples with abnormal C3 levels in primary NBS. Multiple Reaction Monitoring (MRM) mode at m/z 117→73 for MMA and m/z 120→75 for MMA(IS) and isotopic dilution approach was followed for quantification. Results: Intra-assay and inter-assay precision and accuracy was determined at two different levels of MMA (LQC≅2.0 μmol/L and HQC≅10.0 μmol/L), respectively. The Coefficient of Variation (%) for intraday precision ranged between 5.27% to 8.9%. Similarly, for interday it ranged from 4.99% to 9.93%. The average accuracy (%) also falls within (105.4% and 106.1%) for interday and (105.9% and 106.7%) for intraday assay. Stability for samples during storage at different temperature i.e., (fresh, 2-8°C and -20°C) showed long term stability at -20°C storage. The assay was linear over a calibration range of (0.5 to 20.00 μmol/L). Conclusion: The outcome of the present data offers the confidence and reliability in the possible utility of this method for the definitive diagnosis and follows-up of MMA patients.

Methyl transfer reactions catalyzed by cobalamin-dependent enzymes: Insight from molecular docking

Methyl transfer reactions, mediated by methyltransferases (MeTrs), such as methionine synthase (MetH) or monomethylamine: CoM (MtmBC), constitute one of the most important classes of vitamin B12-dependent reactions. The challenge in exploring the catalytic function of MeTrs is related to their modular structure. From the crystallographic point of view, the structure of each subunit has been determined, but there is a lack of understanding of how each subunit interacts with each other. So far, theoretical studies of methyl group transfer were carried out for the structural models of the active site of each subunit. However, those studies do not include the effect of the enzymatic environment, which is crucial for a comprehensive understanding of enzyme-mediated methyl transfer reactions. Herein, to explore how two subunits interact with each other and how the methyl transfer reaction is catalyzed by MeTrs, molecular docking of the functional units of MetH and MtmBC was carried out. Along with the interactions of the functional units, the reaction coordinates, including the Co–C bond distance for methylation of cob(I)alamin (CoICbl) and the C–S bond distance in demethylation reaction of cob(III)alamin (CoIIICbl), were considered. The functional groups should be arranged so that there is an appropriate distance to transfer a methyl group and present results indicate that steric interactions can limit the number of potential arrangements. This calls into question the possibility of SN2-type mechanism previously proposed for MeTrs. Further, it leads to the conclusion that the methyl transfer reaction involves some spatial changes of modules suggesting an alternate radical-based pathway for MeTrs-mediated methyl transfer reactions. The calculations also showed that changes in torsion angles induce a change in reaction coordinates, namely Co–C and C–S bond distances, for the methylation and demethylation reactions catalyzed both by MetH and MtmBC.

Utilization of Cobalamin is Ubiquitous in Early-Branching Fungal Phyla

Cobalamin is a cofactor present in essential metabolic pathways in animals and one of the water-soluble vitamins. It is a complex compound synthesized solely by prokaryotes. Cobalamin dependence is scattered across the tree of life. In particular, fungi and plants were deemed devoid of cobalamin. We demonstrate that cobalamin is utilized by all fungal lineages, except for Dikarya. This observation is supported by the genomic presence of both B12 dependent enzymes and cobalamin modifying enzymes. Moreover, the genes identified are actively transcribed in many taxa. Most fungal cobalamin dependent enzymes and cobalamin metabolism proteins are highly similar to their animal homologs. Phylogenetic analyses support a scenario of vertical inheritance of the cobalamin trait with several losses. Cobalamin usage was probably lost in Mucorinae and at the base of Dikarya which groups most of the model organisms which hindered B12-dependent metabolism discovery in fungi. Our results indicate that cobalamin dependence was a widely distributed trait at least in Opisthokonta, across diverse microbial eukaryotes and likely in the LECA.

Biological Activity of Pseudovitamin B12 on Cobalamin-Dependent Methylmalonyl-CoA Mutase and Methionine Synthase in Mammalian Cultured COS-7 Cells.

Adenyl cobamide (commonly known as pseudovitamin B12) is synthesized by intestinal bacteria or ingested from edible cyanobacteria. The effect of pseudovitamin B12 on the activities of cobalamin-dependent enzymes in mammalian cells has not been studied well. This study was conducted to investigate the effects of pseudovitamin B12 on the activities of the mammalian vitamin B12-dependent enzymes methionine synthase and methylmalonyl-CoA mutase in cultured mammalian COS-7 cells to determine whether pseudovitamin B12 functions as an inhibitor or a cofactor of these enzymes. Although the hydoroxo form of pseudovitamin B12 functions as a coenzyme for methionine synthase in cultured cells, pseudovitamin B12 does not activate the translation of methionine synthase, unlike the hydroxo form of vitamin B12 does. In the second enzymatic reaction, the adenosyl form of pseudovitamin B12 did not function as a coenzyme or an inhibitor of methylmalonyl-CoA mutase. Experiments on the cellular uptake were conducted with human transcobalamin II and suggested that treatment with a substantial amount of pseudovitamin B12 might inhibit transcobalamin II-mediated absorption of a physiological trace concentration of vitamin B12 present in the medium.

Naturally occurring cobalamin (B12) analogs can function as cofactors for human methylmalonyl-CoA mutase

Cobalamin, commonly known as vitamin B12, is an essential micronutrient for humans because of its role as an enzyme cofactor. Cobalamin is one of over a dozen structurally related compounds – cobamides – that are found in certain foods and are produced by microorganisms in the human gut. Very little is known about how different cobamides affect B12-dependent metabolism in human cells. Here, we test in vitro how diverse cobamide cofactors affect the function of methylmalonyl-CoA mutase (MMUT), one of two cobalamin-dependent enzymes in humans. We find that, although cobalamin is the most effective cofactor for MMUT, multiple cobamides support MMUT function with differences in binding affinity (Kd), binding kinetics (kon), and concentration dependence during catalysis (KM, app). Additionally, we find that six disease-associated MMUT variants that cause cobalamin-responsive impairments in enzymatic activity also respond to other cobamides, with the extent of catalytic rescue dependent on the identity of the cobamide. Our studies challenge the exclusive focus on cobalamin in the context of human physiology, indicate that diverse cobamides can support the function of a human enzyme, and suggest future directions that will improve our understanding of the roles of different cobamides in human biology.

Retroconversion of estrogens into androgens by bacteria via a cobalamin-mediated methylation.

Steroid estrogens modulate physiology and development of vertebrates. Conversion of C19 androgens into C18 estrogens is thought to be an irreversible reaction. Here, we report a denitrifying Denitratisoma sp. strain DHT3 capable of catabolizing estrogens or androgens anaerobically. Strain DHT3 genome contains a polycistronic gene cluster, emtABCD, differentially transcribed under estrogen-fed conditions and predicted to encode a cobalamin-dependent methyltransferase system conserved among estrogen-utilizing anaerobes; an emtA-disrupted DHT3 derivative could catabolize androgens but not estrogens. These data, along with the observed androgen production in estrogen-fed strain DHT3 cultures, suggested the occurrence of a cobalamin-dependent estrogen methylation to form androgens. Consistently, the estrogen conversion into androgens in strain DHT3 cell extracts requires methylcobalamin and is inhibited by propyl iodide, a specific inhibitor of cobalamin-dependent enzymes. The identification of the cobalamin-dependent estrogen methylation thus represents an unprecedented metabolic link between cobalamin and steroid metabolism and suggests that retroconversion of estrogens into androgens occurs in the biosphere.

Metagenomic and chemical characterization of soil cobalamin production

Cobalamin (vitamin B12) is an essential enzyme cofactor for most branches of life. Despite the potential importance of this cofactor for soil microbial communities, the producers and consumers of cobalamin in terrestrial environments are still unknown. Here we provide the first metagenome-based assessment of soil cobalamin-producing bacteria and archaea, quantifying and classifying genes encoding proteins for cobalamin biosynthesis, transport, remodeling, and dependency in 155 soil metagenomes with profile hidden Markov models. We also measured several forms of cobalamin (CN-, Me-, OH-, Ado-B12) and the cobalamin lower ligand (5,6-dimethylbenzimidazole; DMB) in 40 diverse soil samples. Metagenomic analysis revealed that less than 10% of soil bacteria and archaea encode the genetic potential for de novo synthesis of this important enzyme cofactor. Predominant soil cobalamin producers were associated with the Proteobacteria, Actinobacteria, Firmicutes, Nitrospirae, and Thaumarchaeota. In contrast, a much larger proportion of abundant soil genera lacked cobalamin synthesis genes and instead were associated with gene sequences encoding cobalamin transport and cobalamin-dependent enzymes. The enrichment of DMB and corresponding DMB synthesis genes, relative to corrin ring synthesis genes, suggests an important role for cobalamin remodelers in terrestrial habitats. Together, our results indicate that microbial cobalamin production and repair serve as keystone functions that are significantly correlated with microbial community size, diversity, and biogeochemistry of terrestrial ecosystems.

Impact of Cyanocobalamin and Methylcobalamin on Inflammatory Bowel Disease and the Intestinal Microbiota Composition.

Patients with inflammatory bowel disease (IBD) are usually advised to supplement various types of vitamin B12, because vitamin B12 is generally absorbed in the colon. Thus, in the current study, the influence of cyanocobalamin (CNCBL) or methylcobalamin (MECBL) ingestion on IBD symptoms will be investigated. Then, whether and how the application of various cobalamins would modify the taxonomic and functional composition of the gut microbiome in mice will be examined carefully. Dextran-sulfate-sodium-induced IBD mice were treated with MECBL or CNCBL; disease activity index (DAI) scores and intestinal inflammatory conditions of mice were evaluated. Fecal samples were collected; microbiota composition was determined with a 16s rRNA analysis; functional profiles were predicted by phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt); and short-chain fatty acids were measured. The consequence of higher relative abundances of Enterobacteriaceae and isomeric short-chain fatty acids by cobalamin treatment revealed that a high concentration of CNCBL but not MECBL supplementation obviously aggravated IBD. A microbial ecosystem rich in Escherichia/ Shigella and low in Lactobacillus, Blautia, and Clostridium XVIII was observed in IBD mice after a high concentration of CNCBL supplementation. In cobalamin-dependent enzymes, CNCBL was more efficient in the adenosylcobalamin system than MECBL and vice versa in the MECBL system. The distinct effects of various cobalamins were associated with the distribution and efficiency of vitamin-B12-dependent riboswitches. CNCBL had a strong inhibitory effect on all riboswitches, especially on btuB and pocR riboswitches from Enterobacteriaceae. CNCBL aggravated IBD via enhancing the proportion of Enterobacteriaceae organisms through riboswitch and enzyme systems. The present study provides a critical reference for offering a suitable amount and type of cobalamin during a symbiotic condition.

Purifying Selective Pressure Suggests the Functionality of a Vitamin B12 Biosynthesis Pathway in a Global Population of Mycobacterium tuberculosis.

Mycobacterium tuberculosis is one of the deadliest and most challenging pathogens to study in current microbiological research. One of the issues that remains to be resolved is the importance of cobalamin in the metabolism of M. tuberculosis. The functionality of a vitamin B12 biosynthesis pathway in M. tuberculosis is under dispute, and the ability of this pathogen to scavenge vitamin B12 from the host is unknown. Here, we quantified the ratios of nonsynonymous and synonymous nucleotide substitution rates (dN/dS) in the genes involved in vitamin B12 biosynthesis and transport and in genes encoding cobalamin-dependent enzymes in nearly four thousand strains of M. tuberculosis. We showed that purifying selection is the dominant force acting on cobalamin-related genes at the levels of individual codons, genes and groups of genes. We conclude that cobalamin-related genes may not be essential but are adaptive for M. tuberculosis in clinical settings. Furthermore, the cobalamin biosynthesis pathway is likely to be functional in this species.

Cobalt tetradehydrocorrins coordinated by imidazolate-like histidine in the heme pocket of horseradish peroxidase.

Horseradish peroxidase was reconstituted with cobalt tetradehydrocorrin, rHRP(Co(TDHC)), as a structural analog of cobalamin coordinated with an imidazolate-like His residue, which is generally seen in native enzymes. In contrast to the previously reported cobalt tetradehydrocorrin-reconstituted myoglobin, rMb(Co(TDHC)), the HRP matrix was expected to provide strong axial ligation by His170 which has imidazolate character. rHRP(CoII(TDHC)) was characterized by EPR and its reaction with reductants indicates a negative shift of its redox potential compared to rMb(Co(TDHC)). Furthermore, aqua- and CN-forms of Co(III) state were prepared. The former species was obtained by oxidation of rHRP(CoII(TDHC)) with K3[Fe(CN)6]. The cyanide-coordinated Co(III) species in the latter was prepared by ligand exchange of rHRP(CoIII(OH)(TDHC)) with exogenous cyanide upon addition of KCN. The 13C NMR chemical shift of cyanide in rHRP(CoIII(CN)(TDHC)) was determined to be 121.8ppm. IR measurements show that the cyanide of rHRP(CoIII(CN)(TDHC)) has a stretching frequency peak at 2144cm-1. The 13C NMR and IR measurements indicate strong coordination of cyanide to CoIII(TDHC) relative to rMb(CoIII(CN)(TDHC)). Thus, the extent of π-back donation from the cobalt ion to the cyanide ion is relatively high in rHRP(CoIII(CN)(TDHC)). The pK 1/2 values of rHRP(CoIII(OH)(TDHC)) and rHRP(CoIII(CN)(TDHC)) are the same (pK 1/2=3.2) as determined by a pH titration experiment, indicating that cyanide ligation does not affect Co-His ligation, whereas cyanide ligation weakens the Co-His ligation in rMb(CoIII(CN)(TDHC)). Taken together, these results indicate that HRP reconstituted with cobalt tetradehydrocorrin is a suitable cobalamin-dependent enzyme model with imidazolate-like His residue.

A common mechanism for coenzyme cobalamin-dependent reductive dehalogenases.

Distinct mechanisms have been proposed for the biological dehalogenation catalyzed by cobalamin-dependent enzymes, with two recent crystallographic studies suggesting different mechanisms based on the observed interaction between the organohalide substrate and cobalamin. In one case, involving an aromatic dibromide substrate in NpRdhA, a novel CoII-Br interaction was observed using EPR, suggesting a mechanism involving a [CoXR] adduct. However, in the case of trichloroethylene in PceA, a significantly longer Co-Cl distance was observed in X-ray crystal structures, suggesting a dissociative electron transfer mechanism. Subsequent DFT models of these reactions have not reproduced these differences in binding modes. Here, we have performed molecular docking and DFT calculations to investigate and compare the interaction between different organohalides and cobalamin in both NpRdhA and PceA. In each case, despite differences in binding in the CoII state, the reaction likely proceeds via formation of a [CoXR] adduct in the CoI state that weakens the breaking carbon-halide bond, suggesting this could be a general mechanism for cobalamin-dependent dehalogenation.