Direct Cobamide Remodeling via Additional Function of Cobamide Biosynthesis Protein CobS from Vibrio cholerae.

Vitamin B12 belongs to a family of structurally diverse cofactors with over a dozen natural analogs, collectively referred to as cobamides. Most bacteria encode cobamide-dependent enzymes, many of which can only utilize a subset of cobamide analogs. Some bacteria employ a mechanism called cobamide remodeling, a process in which cobamides are converted into other analogs to ensure that compatible cobamides are available in the cell. Here, we characterize an additional pathway for cobamide remodeling that is distinct from the previously characterized ones. Cobamide synthase (CobS) is an enzyme required for cobamide biosynthesis that attaches the lower ligand moiety in which the base varies between analogs. In a heterologous model system, we previously showed that Vibrio cholerae CobS (VcCobS) unexpectedly conferred remodeling activity in addition to performing the known cobamide biosynthesis reaction. Here, we show that additional Vibrio species perform the same remodeling reaction, and we further characterize VcCobS-mediated remodeling using bacterial genetics and in vitro assays. We demonstrate that VcCobS acts upon the cobamide pseudocobalamin directly to remodel it, a mechanism which differs from the known remodeling pathways in which cobamides are first cleaved into biosynthetic intermediates. This suggests that some CobS homologs have the additional function of cobamide remodeling, and we propose the term "direct remodeling" for this process. This characterization of yet another pathway for remodeling suggests that cobamide profiles are highly dynamic in polymicrobial environments, with remodeling pathways conferring a competitive advantage. IMPORTANCE Cobamides are widespread cofactors that mediate metabolic interactions in complex microbial communities. Few studies directly examine cobamide profiles, but several have shown that mammalian gastrointestinal tracts are rich in cobamide analogs. Studies of intestinal bacteria, including beneficial commensals and pathogens, show variation in the ability to produce and utilize different cobamides. Some bacteria can convert imported cobamides into compatible analogs in a process called remodeling. Recent discoveries of additional cobamide remodeling pathways, including this work, suggest that remodeling is an important factor in cobamide dynamics. Characterization of such pathways is critical in understanding cobamide flux and nutrient cross-feeding in polymicrobial communities, and it facilitates the establishment of microbiome manipulation strategies via modulation of cobamide profiles.

Insights into the Relationship between Cobamide Synthase and the Cell Membrane.

Cobamides are cobalt-containing cyclic tetrapyrroles used by cells from all domains of life but only produced de novo by some bacteria and archaea. The "late steps" of the adenosylcobamide biosynthetic pathway are responsible for the assembly of the nucleotide loop and are required during de novo synthesis and precursor salvaging. These steps are characterized by activation of the corrin ring and lower ligand base, condensation of the activated precursors to adenosylcobamide phosphate, and removal of the phosphate, yielding a complete adenosylcobamide molecule. The condensation of the activated corrin ring and lower ligand base is performed by an integral membrane protein, cobamide (5' phosphate) synthase (CobS), and represents an important convergence of two pathways necessary for nucleotide loop assembly. Interestingly, membrane association of this penultimate step is conserved among all cobamide producers, yet the physiological relevance of this association is not known. Here, we present the purification and biochemical characterization of the CobS enzyme of the enterobacterium Salmonella enterica subsp. enterica serovar Typhimurium strain LT2, investigate its association with liposomes, and quantify the effect of the lipid bilayer on its enzymatic activity and substrate affinity. We report a purification scheme that yields pure CobS protein, allowing in vitro functional analysis. Additionally, we report a method for liposome reconstitution of CobS, allowing for physiologically relevant studies of this inner membrane protein in a phospholipid bilayer. In vitro and in vivo data reported here expand our understanding of CobS and the implications of membrane-associated adenosylcobamide biosynthesis.IMPORTANCESalmonella is a human pathogen of worldwide importance, and coenzyme B12 is critical for the pathogenic lifestyle of this bacterium. The importance of the work reported here lies on the improvements to the methodology used to isolate cobamide synthase, a polytopic integral membrane protein that catalyzes the penultimate step of coenzyme B12 biosynthesis. This advance is an important step in the analysis of the proposed multienzyme complex responsible for the assembly of the nucleotide loop during de novo coenzyme B12 biosynthesis and for the assimilation of incomplete corrinoids from the environment. We proposed that cobamide synthase is likely localized to the cell membrane of every coenzyme B12-producing bacterium and archaeum sequenced to date. The new knowledge of cobamide synthase advances our understanding of the functionality of the enzyme in the context of the lipid bilayer and sets the foundation for the functional-structural analysis of the aforementioned multienzyme complex.

CobT and BzaC catalyze the regiospecific activation and methylation of the 5-hydroxybenzimidazole lower ligand in anaerobic cobamide biosynthesis.

Vitamin B12 and other cobamides are essential cofactors required by many organisms and are synthesized by a subset of prokaryotes via distinct aerobic and anaerobic routes. The anaerobic biosynthesis of 5,6-dimethylbenzimidazole (DMB), the lower ligand of vitamin B12, involves five reactions catalyzed by the bza operon gene products, namely the hydroxybenzimidazole synthase BzaAB/BzaF, phosphoribosyltransferase CobT, and three methyltransferases, BzaC, BzaD, and BzaE, that conduct three distinct methylation steps. Of these, the methyltransferases that contribute to benzimidazole lower ligand diversity in cobamides remain to be characterized, and the precise role of the bza operon protein CobT is unclear. In this study, we used the bza operon from the anaerobic bacterium Moorella thermoacetica (comprising bzaA-bzaB-cobT-bzaC) to examine the role of CobT and investigate the activity of the first methyltransferase, BzaC. We studied the phosphoribosylation catalyzed by MtCobT and found that it regiospecifically activates 5-hydroxybenzimidazole (5-OHBza) to form the 5-OHBza-ribotide (5-OHBza-RP) isomer as the sole product. Next, we characterized the domains of MtBzaC and reconstituted its methyltransferase activity with the predicted substrate 5-OHBza and with two alternative substrates, the MtCobT product 5-OHBza-RP and its riboside derivative 5-OHBza-R. Unexpectedly, we found that 5-OHBza-R is the most favored MtBzaC substrate. Our results collectively explain the long-standing observation that the attachment of the lower ligand in anaerobic cobamide biosynthesis is regiospecific. In conclusion, we validate MtBzaC as a SAM:hydroxybenzimidazole-riboside methyltransferase (HBIR-OMT). Finally, we propose a new pathway for the synthesis and activation of the benzimidazolyl lower ligand in anaerobic cobamide biosynthesis.

Structural and functional analysis of an l-serine O-phosphate decarboxylase involved in norcobamide biosynthesis.

Structural diversity of natural cobamides (Cbas, B12 vitamers) is limited to the nucleotide loop. The loop is connected to the cobalt-containing corrin ring via an (R)-1-aminopropan-2-ol O-2-phosphate (AP-P) linker moiety. AP-P is produced by the l-threonine O-3-phosphate (l-Thr-P) decarboxylase CobD. Here, the CobD homolog SMUL_1544 of the organohalide-respiring epsilonproteobacterium Sulfurospirillum multivorans was characterized as a decarboxylase that produces ethanolamine O-phosphate (EA-P) from l-serine O-phosphate (l-Ser-P). EA-P is assumed to serve as precursor of the linker moiety of norcobamides that function as cofactors in the respiratory reductive dehalogenase. SMUL_1544 (SmCobD) is a pyridoxal-5'-phosphate (PLP)-containing enzyme. The structural analysis of the SmCobD apoprotein combined with the characterization of truncated mutant proteins uncovered a role of the SmCobD N-terminus in efficient l-Ser-P conversion.

A New Class of Phosphoribosyltransferases Involved in Cobamide Biosynthesis Is Found in Methanogenic Archaea and Cyanobacteria.

Cobamides are coenzymes used by cells from all domains of life but made de novo by only some bacteria and archaea. The last steps of the cobamide biosynthetic pathway activate the corrin ring and the lower ligand base, condense the activated intermediates, and dephosphorylate the product prior to the release of the biologically active coenzyme. In bacteria, a phosphoribosyltransferase (PRTase) enyzme activates the base into its α-mononucleotide. The enzyme from Salmonella enterica ( SeCobT) has been extensively biochemically and structurally characterized. The crystal structure of the putative PRTase from the archaeum Methanocaldococcus jannaschii ( MjCobT) is known, but its function has not been validated. Here we report the in vivo and in vitro characterization of MjCobT. In vivo, in vitro, and phylogenetic data reported here show that MjCobT belongs to a new class of NaMN-dependent PRTases. We also show that the Synechococcus sp. WH7803 CobT protein has PRTase activity in vivo. Lastly, results of isothermal titration calorimetry and analytical ultracentrifugation analysis show that the biologically active form of MjCobT is a dimer, not a trimer, as suggested by its crystal structure.

Uneven distribution of cobamide biosynthesis and dependence in bacteria predicted by comparative genomics.

The vitamin B12 family of cofactors known as cobamides are essential for a variety of microbial metabolisms. We used comparative genomics of 11,000 bacterial species to analyze the extent and distribution of cobamide production and use across bacteria. We find that 86% of bacteria in this data set have at least one of 15 cobamide-dependent enzyme families, but only 37% are predicted to synthesize cobamides de novo. The distribution of cobamide biosynthesis and use vary at the phylum level. While 57% of Actinobacteria are predicted to biosynthesize cobamides, only 0.6% of Bacteroidetes have the complete pathway, yet 96% of species in this phylum have cobamide-dependent enzymes. The form of cobamide produced by the bacteria could be predicted for 58% of cobamide-producing species, based on the presence of signature lower ligand biosynthesis and attachment genes. Our predictions also revealed that 17% of bacteria have partial biosynthetic pathways, yet have the potential to salvage cobamide precursors. Bacteria with a partial cobamide biosynthesis pathway include those in a newly defined, experimentally verified category of bacteria lacking the first step in the biosynthesis pathway. These predictions highlight the importance of cobamide and cobamide precursor salvaging as examples of nutritional dependencies in bacteria.

Guided cobamide biosynthesis for heterologous production of reductive dehalogenases.

Cobamides (Cbas) are essential cofactors of reductive dehalogenases (RDases) in organohalide-respiring bacteria (OHRB). Changes in the Cba structure can influence RDase function. Here, we report on the cofactor versatility or selectivity of Desulfitobacterium RDases produced either in the native organism or heterologously. The susceptibility of Desulfitobacterium hafniense strain DCB-2 to guided Cba biosynthesis (i.e. incorporation of exogenous Cba lower ligand base precursors) was analysed. Exogenous benzimidazoles, azabenzimidazoles and 4,5-dimethylimidazole were incorporated by the organism into Cbas. When the type of Cba changed, no effect on the turnover rate of the 3-chloro-4-hydroxy-phenylacetate-converting enzyme RdhA6 and the 3,5-dichlorophenol-dehalogenating enzyme RdhA3 was observed. The impact of the amendment of Cba lower ligand precursors on RDase function was also investigated in Shimwellia blattae, the Cba producer used for the heterologous production of Desulfitobacterium RDases. The recombinant tetrachloroethene RDase (PceAY51 ) appeared to be non-selective towards different Cbas. However, the functional production of the 1,2-dichloroethane-dihaloeliminating enzyme (DcaA) of Desulfitobacterium dichloroeliminans was completely prevented in cells producing 5,6-dimethylbenzimidazolyl-Cba, but substantially enhanced in cells that incorporated 5-methoxybenzimidazole into the Cba cofactor. The results of the study indicate the utilization of a range of different Cbas by Desulfitobacterium RDases with selected representatives apparently preferring distinct Cbas.

Novel Hybrid Input Part Using Riboswitch and Transcriptional Repressor for Signal Inverting Amplifier.

Genetic circuits are composed of input, logic, and output parts. Construction of complex circuits for practical applications requires numerous tunable genetic parts. However, the limited diversity and complicated tuning methods used for the input parts hinders the scalability of genetic circuits. Therefore, a new type of input part is required that responds to diverse signals and enables easy tuning. Here, we developed RNA-protein hybrid input parts that combine a riboswitch and orthogonal transcriptional repressors. The hybrid inputs successfully regulated the transcription of an output in response to the input signal detected by the riboswitch and resulted in signal inversion because of the expression of transcriptional repressors. Dose-response parameters including fold-change and half-maximal effective concentration were easily modulated and amplified simply by changing the promoter strength. Furthermore, the hybrid input detected both exogenous and endogenous signals, indicating potential applications in metabolite sensing. This hybrid input part could be highly extensible considering the rich variety of components.

Purinyl-cobamide is a native prosthetic group of reductive dehalogenases.

Cobamides such as vitamin B12 are structurally conserved, cobalt-containing tetrapyrrole biomolecules that have essential biochemical functions in all domains of life. In organohalide respiration, a vital biological process for the global cycling of natural and anthropogenic organohalogens, cobamides are the requisite prosthetic groups for carbon-halogen bond-cleaving reductive dehalogenases. This study reports the biosynthesis of a new cobamide with unsubstituted purine as the lower base and assigns unsubstituted purine a biological function by demonstrating that Coα-purinyl-cobamide (purinyl-Cba) is the native prosthetic group in catalytically active tetrachloroethene reductive dehalogenases of Desulfitobacterium hafniense. Cobamides featuring different lower bases are not functionally equivalent, and purinyl-Cba elicits different physiological responses in corrinoid-auxotrophic, organohalide-respiring bacteria. Given that cobamide-dependent enzymes catalyze key steps in essential metabolic pathways, the discovery of a novel cobamide structure and the realization that lower bases can effectively modulate enzyme activities generate opportunities to manipulate functionalities of microbiomes.

The cause of multiple sclerosis is autoimmune attack of adenosyltransferase thereby limiting adenosylcobalamin production.

The pathogenesis of multiple sclerosis (MS) begins with an infection by a bacterium from the class of bacteria that produce and utilize adenosylcobalamin (AdoCbl) and possess an adenosyl transferase enzyme (ATR); these bacteria are the exogenous antigens that cause MS. Human ATR is homologous to bacterial ATR and B cells produce anti-ATR antibodies as an autoimmune response thereby reducing the concentration of ATR and thus limiting production of AdoCbl, one of the two bioactive forms of vitamin B12. The next step in MS pathogenesis is a period of subclinical AdoCbl deficiency over a period of many years resulting in production of odd-carbon-number fatty acids that are incorporated into myelin rendering it antigenic. The next step in MS pathogenesis is breach of the blood brain barrier thereby introducing leukocytes into the brain's blood supply resulting in T cell attack of antigenic myelin. All epidemiological clusters are regions wherein the major agricultural products are legumes that produce a high percentage of odd-carbon-number fatty acids and contain symbiotic rhizobia type bacteria in root nodules and in the soil. This novel etiological hypothesis is called "multiple sclerosis due to adenosylcobalamin deficiency" (MS-AdoCbl). Creation of realistic animal models based on the MS-AdoCbl hypothesis is presented. Methods for testing predictions made by the MS-AdoCbl hypothesis are described.

Coenzyme B12 synthesis as a baseline to study metabolite contribution of animal microbiota.

Microbial communities thrive in a number of environments. Exploration of their microbiomes - their global genome - may reveal metabolic features that contribute to the development and welfare of their hosts, or chemical cleansing of environments. Yet we often lack final demonstration of their causal role in features of interest. The reason is that we do not have proper baselines that we could use to monitor how microbiota cope with key metabolites in the hosting environment. Here, focusing on animal gut microbiota, we describe the fate of cobalamins - metabolites of the B12 coenzyme family - that are essential for animals but synthesized only by prokaryotes. Microbiota produce the vitamin used in a variety of animals (and in algae). Coprophagy plays a role in its management. For coprophobic man, preliminary observations suggest that the gut microbial production of vitamin B12 plays only a limited role. By contrast, the vitamin is key for structuring microbiota. This implies that it is freely available in the environment. This can only result from lysis of the microbes that make it. A consequence for biotechnology applications is that, if valuable for their host, B12-producing microbes should be sensitive to bacteriophages and colicins, or make spores.

The organohalide-respiring bacterium Sulfurospirillum multivorans: a natural source for unusual cobamides.

Cobamides ('complete' corrinoids) are essential for organohalide-respiring bacteria because they act as cofactors of reductive dehalogenases (RDases). RDases are the key enzymes in organohalide respiration, a process relevant for environmental remediation. More than a decade ago, the unusual norpseudo-B12 was identified as cofactor of the tetrachloroethene RDase (PceA) purified from the epsilonproteobacterium Sulfurospirillum multivorans. Since then, the question was raised whether or not the production of the uncommon cobamide is a specific adaptation to the requirements of PceA. Recently, efforts were made to unravel variations in the cobamide biosynthetic pathway, which lead to the production of the structurally unique norpseudo-B12. The acquisition of genomic and proteomic data together with structural analyses of PceA provided insights into norcobamide formation and utilization. By the use of guided biosynthesis, S. multivorans was shown to be an effective cobamide producer capable of generating unusual norcobamides either functional or non-functional as cofactors of PceA. The organism turned out to be a suitable tool for testing the impact of cobamide structure on enzyme function. The results summarized here highlight S. multivorans in particular and the organohalide-respiring bacteria in general as a resource for new discoveries on cobamide diversity and utilization.

Salmonella enterica synthesizes 5,6-dimethylbenzimidazolyl-(DMB)-α-riboside. Why some Firmicutes do not require the canonical DMB activation system to synthesize adenosylcobalamin.

5,6-Dimethylbenzimidazolyl-(DMB)-α-ribotide [α-ribazole-5'-phosphate (α-RP)] is an intermediate in the biosynthesis of adenosylcobalamin (AdoCbl) in many prokaryotes. In such microbes, α-RP is synthesized by nicotinate mononucleotide (NaMN):DMB phosphoribosyltransferases (CobT in Salmonella enterica), in a reaction that is considered to be the canonical step for the activation of the base of the nucleotide present in adenosylcobamides. Some Firmicutes lack CobT-type enzymes but have a two-protein system comprised of a transporter (i.e., CblT) and a kinase (i.e., CblS) that can salvage exogenous α-ribazole (α-R) from the environment using CblT to take up α-R, followed by α-R phosphorylation by CblS. We report that Geobacillus kaustophilus CblT and CblS proteins restore α-RP synthesis in S. enterica lacking the CobT enzyme. We also show that a S. enterica cobT strain that synthesizes GkCblS ectopically makes only AdoCbl, even under growth conditions where the synthesis of pseudoCbl is favored. Our results indicate that S. enterica synthesizes α-R, a metabolite that had not been detected in this bacterium and that GkCblS has a strong preference for DMB-ribose over adenine-ribose as substrate. We propose that in some Firmicutes DMB is activated to α-RP via α-R using an as-yet-unknown route to convert DMB to α-R and CblS to convert α-R to α-RP.

The SMUL_1544 Gene Product Governs Norcobamide Biosynthesis in the Tetrachloroethene-Respiring Bacterium Sulfurospirillum multivorans.

UNLABELLED: The tetrachloroethene (PCE)-respiring bacterium Sulfurospirillum multivorans produces a unique cobamide, namely, norpseudo-B12, which, in comparison to other cobamides, e.g., cobalamin and pseudo-B12, lacks the methyl group in the linker moiety of the nucleotide loop. In this study, the protein SMUL_1544 was shown to be responsible for the formation of the unusual linker moiety, which is most probably derived from ethanolamine-phosphate (EA-P) as the precursor. The product of the SMUL_1544 gene successfully complemented a Salmonella enterica ΔcobD mutant. The cobD gene encodes an l-threonine-O-3-phosphate (l-Thr-P) decarboxylase responsible for the synthesis of (R)-1-aminopropan-2-ol O-2-phosphate (AP-P), required specifically for cobamide biosynthesis. When SMUL_1544 was produced in the heterologous host lacking CobD, norpseudo-B12 was formed, which pointed toward the formation of EA-P rather than AP-P. Guided cobamide biosynthesis experiments with minimal medium supplemented with l-Thr-P supported cobamide biosynthesis in S. enterica producing SMUL_1544 or S. multivorans Under these conditions, both microorganisms synthesized pseudo-B12 This observation indicated a flexibility in the SMUL_1544 substrate spectrum. From the formation of catalytically active PCE reductive dehalogenase (PceA) in S. multivorans cells producing pseudo-B12, a compatibility of the respiratory enzyme with the cofactor was deduced. This result might indicate a structural flexibility of PceA in cobamide binding. Feeding of l-[3-(13)C]serine to cultures of S. multivorans resulted in isotope labeling of the norpseudo-B12 linker moiety, which strongly supports the hypothesis of EA-P formation from l-serine-O-phosphate (l-Ser-P) in this organism. IMPORTANCE: The identification of the gene product SMUL_1544 as a putative l-Ser-P decarboxylase involved in norcobamide biosynthesis in S. multivorans adds a novel module to the assembly line of cobamides (complete corrinoids) in prokaryotes. Selected cobamide-containing enzymes (e.g., reductive dehalogenases) showed specificity for their cobamide cofactors. It has recently been proposed that the structure of the linker moiety of norpseudo-B12 and the mode of binding of the EA-P linker to the PceA enzyme reflect the high specificity of the enzyme for its cofactor. Data reported herein do not support this idea. In fact, norpseudo-B12 was functional in the cobamide-dependent methionine biosynthesis of S. enterica, raising questions about the role of norcobamides in nature.

Fermentative production of 1-propanol from d-glucose, l-rhamnose and glycerol using recombinant Escherichia coli.

Fermentative production of 1-propanol, which is one of the promising precursors of polypropylene production, from d-glucose, l-rhamnose and glycerol using metabolically engineered Escherichia coli was examined. To confer the ability to produce 1-propanol from 1,2-propanediol (1,2-PD) in recombinant E. coli, a part of the pdu regulon including the diol dehydratase and the propanol dehydrogenase genes together with the adenosylcobalamin (AdoCbl) regeneration enzyme genes of Klebsiella pneumoniae was cloned, and an expression vector for these genes (pRSF_pduCDEGHOQS) was constructed. Recombinant E. coli harboring pRSF_pduCDEGHOQS with 1,2-PD synthetic pathway (pKK_mde) genes, which was constructed in our previous report (Urano et al., Appl. Microbiol. Biotechnol., 99, 2001-2008, 2015), produced 16.1 mM of 1-propanol from d-glucose with a molar yield of 0.36 mol/mol after 72 h cultivation. 29.9 mM of 1-propanol was formed from l-rhamnose with a molar yield of 0.81 mol/mol using E. coli carrying only pRSF_pduCDEGHOQS. In addition, 1-propanol production from glycerol was achieved by addition of the ATP-dependent dihydroxyacetone kinase gene to E. coli harboring pKK_mde and pRSF_pduCDEGOQS. In all cases, 1-propanol production was achieved by adding only a small amount of AdoCbl.

Multiple congenital anomalies in two boys with mutation in HCFC1 and cobalamin disorder.

The cobalamin type C deficiency is a rare condition that results from impaired biosynthesis of both methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl). Hemizygous mutations of the HCFC1 gene explain the majority of clinically and biologically compatible cblC patients without MMACHC mutations (OMIM 309541). We report a family with two maternal half-brothers with multiple congenital anomalies and HCFC1 gene mutation in the second Kelch domain. Both presented with dysmorphic features (flat profile, cleft lip for one), increased nuchal translucency, prenatal onset microcephaly and hypospadias. Additionally to early onset intractable epilepsy and profound neurocognitive impairment, this familial observation suggests that HCFC1 gene should be considered in boys with midline malformations, even without proven cobalamin C deficiency.

Coenzyme B12 can be produced by engineered Escherichia coli under both anaerobic and aerobic conditions.

Coenzyme B12 (Vitamin B12 ) is one of the most complex biomolecules and an essential cofactor required for the catalytic activity of many enzymes. Pseudomonas denitrificans synthesizes coenzyme B12 in an oxygen-dependent manner using a pathway encoded by more than 25 genes that are located in six different operons. Escherichia coli, a robust and suitable host for metabolic engineering was used to produce coenzyme B12 . These genes were cloned into three compatible plasmids and expressed heterologously in E. coli BL21 (DE3). Real-time PCR, SDS-PAGE analysis and bioassay showed that the recombinant E. coli expressed the coenzyme B12 synthetic genes and successfully produced coenzyme B12 . However, according to the quantitative determination by inductively coupled plasma-mass spectrometry, the amount of coenzyme B12 produced by the recombinant E. coli (0.21 ± 0.02 µg/g cdw) was approximately 13-fold lower than that by P. denitrificans (2.75 ± 0.22 µg/g cdw). Optimization of the culture conditions to improve the production of coenzyme B12 by the recombinant E. coli was successful, and the highest titer (0.65 ± 0.03 µg/g cdw) of coenzyme B12 was obtained. Interestingly, although the synthesis of coenzyme B12 in P. denitrificans is strictly oxygen-dependent, the recombinant E. coli could produce coenzyme B12 under anaerobic conditions.

Functional heterologous production of reductive dehalogenases from Desulfitobacterium hafniense strains.

The anaerobic dehalogenation of organohalides is catalyzed by the reductive dehalogenase (RdhA) enzymes produced in phylogenetically diverse bacteria. These enzymes contain a cobamide cofactor at the active site and two iron-sulfur clusters. In this study, the tetrachloroethene (PCE) reductive dehalogenase (PceA) of the Gram-positive Desulfitobacterium hafniense strain Y51 was produced in a catalytically active form in the nondechlorinating, cobamide-producing bacterium Shimwellia blattae (ATCC 33430), a Gram-negative gammaproteobacterium. The formation of recombinant catalytically active PceA enzyme was significantly enhanced when its dedicated PceT chaperone was coproduced and when 5,6-dimethylbenzimidazole and hydroxocobalamin were added to the S. blattae cultures. The experiments were extended to D. hafniense DCB-2, a reductively dehalogenating bacterium harboring multiple rdhA genes. To elucidate the substrate spectrum of the rdhA3 gene product of this organism, the recombinant enzyme was tested for the conversion of different dichlorophenols (DCP) in crude extracts of an RdhA3-producing S. blattae strain. 3,5-DCP, 2,3-DCP, and 2,4-DCP, but not 2,6-DCP and 3,4-DCP, were reductively dechlorinated by the recombinant RdhA3. In addition, this enzyme dechlorinated PCE to trichloroethene at low rates.

Exogenous 5,6-dimethylbenzimidazole caused production of a non-functional tetrachloroethene reductive dehalogenase in Sulfurospirillum multivorans.

Corrinoid-dependent reductive dehalogenation is mediated by phylogenetically diverse anaerobic bacteria that either synthesize corrinoids de novo or are dependent on corrinoid salvaging from the environment. The tetrachloroethene (PCE) reductive dehalogenase (PceA) of the Gram-negative Epsilonproteobacterium Sulfurospirillum multivorans harbours a norpseudo-B12 as corrinoid cofactor. Norpseudo-B12 differs from coenzyme B12 in the nucleotide loop structure. Adenine instead of 5,6-dimethylbenzimidazole (DMB) serves as lower ligand base of the central cobalt ion, and the nucleotide loop of norpseudo-B12 lacks a methyl group at position 176. In this study, S. multivorans was grown anaerobically with PCE in the presence of DMB. At a DMB concentration of 25 µM, the adenine moiety in the nucleotide loop of norpseudo-B12 was quantitatively replaced by DMB. The formation of the DMB-containing nor-B12 severely affected PCE-dependent growth and the PceA activity. In DMB-treated cells processing of the cytoplasmic PceA precursor was impeded, a result pointing to retarded cofactor incorporation. PceA enriched from cells cultivated with DMB contained nor-B12 . Nor-B12 purified from cells grown in the presence of DMB mediated the abiotic reductive dehalogenation of trichloroacetate to dichloroacetate at a 25-fold lower rate in comparison with norpseudo-B12 , a fact underpinning the relevance of norpseudo-B12 as efficient catalyst for reductive dehalogenation in general.