A New Facet of Vitamin B12: Gene Regulation by Cobalamin-Based Photoreceptors.

Living organisms sense and respond to light, a crucial environmental factor, using photoreceptors, which rely on bound chromophores such as retinal, flavins, or linear tetrapyrroles for light sensing. The discovery of photoreceptors that sense light using 5'-deoxyadenosylcobalamin, a form of vitamin B12 that is best known as an enzyme cofactor, has expanded the number of known photoreceptor families and unveiled a new biological role of this vitamin. The prototype of these B12-dependent photoreceptors, the transcriptional repressor CarH, is widespread in bacteria and mediates light-dependent gene regulation in a photoprotective cellular response. CarH activity as a transcription factor relies on the modulation of its oligomeric state by 5'-deoxyadenosylcobalamin and light. This review surveys current knowledge about these B12-dependent photoreceptors, their distribution and mode of action, and the structural and photochemical basis of how they orchestrate signal transduction and control gene expression.

Ligand cross-feeding resolves bacterial vitamin B12 auxotrophies.

Cobalamin (vitamin B12, herein referred to as B12) is an essential cofactor for most marine prokaryotes and eukaryotes1,2. Synthesized by a limited number of prokaryotes, its scarcity affects microbial interactions and community dynamics2-4. Here we show that two bacterial B12 auxotrophs can salvage different B12 building blocks and cooperate to synthesize B12. A Colwellia sp. synthesizes and releases the activated lower ligand α-ribazole, which is used by another B12 auxotroph, a Roseovarius sp., to produce the corrin ring and synthesize B12. Release of B12 by Roseovarius sp. happens only in co-culture with Colwellia sp. and only coincidently with the induction of a prophage encoded in Roseovarius sp. Subsequent growth of Colwellia sp. in these conditions may be due to the provision of B12 by lysed cells of Roseovarius sp. Further evidence is required to support a causative role for prophage induction in the release of B12. These complex microbial interactions of ligand cross-feeding and joint B12 biosynthesis seem to be widespread in marine pelagic ecosystems. In the western and northern tropical Atlantic Ocean, bacteria predicted to be capable of salvaging cobinamide and synthesizing only the activated lower ligand outnumber B12 producers. These findings add new players to our understanding of B12 supply to auxotrophic microorganisms in the ocean and possibly in other ecosystems.

Riboswitch and small RNAs modulate btuB translation initiation in Escherichia coli and trigger distinct mRNA regulatory mechanisms.

Small RNAs (sRNAs) and riboswitches represent distinct classes of RNA regulators that control gene expression upon sensing metabolic or environmental variations. While sRNAs and riboswitches regulate gene expression by affecting mRNA and protein levels, existing studies have been limited to the characterization of each regulatory system in isolation, suggesting that sRNAs and riboswitches target distinct mRNA populations. We report that the expression of btuB in Escherichia coli, which is regulated by an adenosylcobalamin (AdoCbl) riboswitch, is also controlled by the small RNAs OmrA and, to a lesser extent, OmrB. Strikingly, we find that the riboswitch and sRNAs reduce mRNA levels through distinct pathways. Our data show that while the riboswitch triggers Rho-dependent transcription termination, sRNAs rely on the degradosome to modulate mRNA levels. Importantly, OmrA pairs with the btuB mRNA through its central region, which is not conserved in OmrB, indicating that these two sRNAs may have specific targets in addition to their common regulon. In contrast to canonical sRNA regulation, we find that OmrA repression of btuB is lost using an mRNA binding-deficient Hfq variant. Together, our study demonstrates that riboswitch and sRNAs modulate btuB expression, providing an example of cis- and trans-acting RNA-based regulatory systems maintaining cellular homeostasis.

Characterization of a Structurally Distinct ATP-Dependent Reactivating Factor of Adenosylcobalamin-Dependent Lysine 5,6-Aminomutase.

Several anaerobic bacterial species, including the Gram-negative oral bacterium Fusobacterium nucleatum, ferment lysine to produce butyrate, acetate, and ammonia. The second step of the metabolic pathway─isomerization of ß-l-lysine to erythro-3,5-diaminohexanoate─is catalyzed by the adenosylcobalamin (AdoCbl) and pyridoxal 5'-phosphate (PLP)-dependent enzyme, lysine 5,6-aminomutase (5,6-LAM). Similar to other AdoCbl-dependent enzymes, 5,6-LAM undergoes mechanism-based inactivation due to loss of the AdoCbl 5'-deoxyadenosyl moiety and oxidation of the cob(II)alamin intermediate to hydroxocob(III)alamin. Herein, we identified kamB and kamC, two genes responsible for ATP-dependent reactivation of 5,6-LAM. KamB and KamC, which are encoded upstream of the genes corresponding to α and ß subunits of 5,6-LAM (kamD and kamE), co-purified following coexpression of the genes in Escherichia coli. KamBC exhibited a basal level of ATP-hydrolyzing activity that was increased 35% in a reaction mixture that facilitated 5,6-LAM turnover with ß-l-lysine or d,l-lysine. Ultraviolet-visible (UV-vis) spectroscopic studies performed under anaerobic conditions revealed that KamBC in the presence of ATP/Mg2+ increased the steady-state concentration of the cob(II)alamin intermediate in the presence of excess ß-l-lysine. Using a coupled UV-visible spectroscopic assay, we show that KamBC is able to reactivate 5,6-LAM through exchange of the damaged hydroxocob(III)alamin for AdoCbl. KamBC is also specific for 5,6-LAM as it had no effect on the rate of substrate-induced inactivation of the homologue, ornithine 4,5-aminomutase. Based on sequence homology, KamBC is structurally distinct from previously characterized B12 chaperones and reactivases, and correspondingly adds to the list of proteins that have evolved to maintain the cellular activity of B12 enzymes.

Structural insight into G-protein chaperone-mediated maturation of a bacterial adenosylcobalamin-dependent mutase.

G-protein metallochaperones are essential for the proper maturation of numerous metalloenzymes. The G-protein chaperone MMAA in humans (MeaB in bacteria) uses GTP hydrolysis to facilitate the delivery of adenosylcobalamin (AdoCbl) to AdoCbl-dependent methylmalonyl-CoA mutase, an essential metabolic enzyme. This G-protein chaperone also facilitates the removal of damaged cobalamin (Cbl) for repair. Although most chaperones are standalone proteins, isobutyryl-CoA mutase fused (IcmF) has a G-protein domain covalently attached to its target mutase. We previously showed that dimeric MeaB undergoes a 180° rotation to reach a state capable of GTP hydrolysis (an active G-protein state), in which so-called switch III residues of one protomer contact the G-nucleotide of the other protomer. However, it was unclear whether other G-protein chaperones also adopted this conformation. Here, we show that the G-protein domain in a fused system forms a similar active conformation, requiring IcmF oligomerization. IcmF oligomerizes both upon Cbl damage and in the presence of the nonhydrolyzable GTP analog, guanosine-5'-[(ß,γ)-methyleno]triphosphate, forming supramolecular complexes observable by mass photometry and EM. Cryo-EM structural analysis reveals that the second protomer of the G-protein intermolecular dimer props open the mutase active site using residues of switch III as a wedge, allowing for AdoCbl insertion or damaged Cbl removal. With the series of structural snapshots now available, we now describe here the molecular basis of G-protein-assisted AdoCbl-dependent mutase maturation, explaining how GTP binding prepares a mutase for cofactor delivery and how GTP hydrolysis allows the mutase to capture the cofactor.

Corrin Ring Modifications Reveal the Chemical and Spatial Requirements for the B12-btuB Riboswitch Interaction.

The btuB riboswitch is a regulatory RNA sequence controlling gene expression of the outer membrane B12 transport protein BtuB by specifically binding coenzyme B12 (AdoCbl) as its natural ligand. The B12 sensing riboswitch class is known to accept various B12 derivatives, leading to a division into two riboswitch subclasses, dependent on the size of the apical ligand. Here we focus on the role of side chains b and e on affinity and proper recognition, i. e. correct structural switch of the btuB RNA, which belongs to the AdoCbl-binding class I. Chemical modification of these side chains disturbs crucial hydrogen bonds and/or electrostatic interactions with the RNA, its effect on both affinity and switching being monitored by in-line probing. Chemical modifications at sidechain b of vitamin B12 show larger effects indicating crucial B12-RNA interactions. When introducing the same modification to AdoCbl the influence of any side-chain modification tested is reduced. This renders the impact of the adenosyl-ligand for B12-btuB riboswitch recognition clearly beyond the known role in affinity.

The Rhodium Analogue of Coenzyme B12 as an Anti-Photoregulatory Ligand Inhibiting Bacterial CarH Photoreceptors.

Coenzyme B12 (AdoCbl; 5'-deoxy-5'-adenosylcobalamin), the quintessential biological organometallic radical catalyst, has a formerly unanticipated, yet extensive, role in photoregulation in bacteria. The light-responsive cobalt-corrin AdoCbl performs this nonenzymatic role by facilitating the assembly of CarH photoreceptors into DNA-binding tetramers in the dark, suppressing gene expression. Conversely, exposure to light triggers the decomposition of this AdoCbl-bound complex by a still elusive photochemical mechanism, activating gene expression. Here, we have examined AdoRhbl, the non-natural rhodium analogue of AdoCbl, as a photostable isostructural surrogate for AdoCbl. We show that AdoRhbl closely emulates AdoCbl in its uptake by bacterial cells and structural functionality as a regulatory ligand for CarH tetramerization, DNA binding, and repressor activity. Remarkably, we find AdoRhbl is photostable even when bound "base-off/His-on" to CarH in vitro and in vivo. Thus, AdoRhbl, an antivitamin B12, also represents an unprecedented anti-photoregulatory ligand, opening a pathway to precisely target biomimetic inhibition of AdoCbl-based photoregulation, with new possibilities for selective antibacterial applications. Computational biomolecular analysis of AdoRhbl binding to CarH yields detailed structural insights into this complex, which suggest that the adenosyl group of photoexcited AdoCbl bound to CarH may specifically undergo a concerted non-radical syn-1,2-elimination mechanism, an aspect not previously considered for this photoreceptor.

Localization and interaction studies of the Salmonella enterica ethanolamine ammonia-lyase (EutBC), its reactivase (EutA), and the EutT corrinoid adenosyltransferase.

Some prokaryotes compartmentalize select metabolic capabilities. Salmonella enterica subspecies enterica serovar Typhimurium LT2 (hereafter S. Typhimurium) catabolizes ethanolamine (EA) within a proteinaceous compartment that we refer to as the ethanolamine utilization (Eut) metabolosome. EA catabolism is initiated by the adenosylcobalamin (AdoCbl)-dependent ethanolamine ammonia-lyase (EAL), which deaminates EA via an adenosyl radical mechanism to yield acetaldehyde plus ammonia. This adenosyl radical can be quenched, requiring the replacement of AdoCbl by the ATP-dependent EutA reactivase. During growth on ethanolamine, S. Typhimurium synthesizes AdoCbl from cobalamin (Cbl) using the ATP:Co(I)rrinoid adenosyltransferase (ACAT) EutT. It is known that EAL localizes to the metabolosome, however, prior to this work, it was unclear where EutA and EutT localized, and whether they interacted with EAL. Here, we provide evidence that EAL, EutA, and EutT localize to the Eut metabolosome, and that EutA interacts directly with EAL. We did not observe interactions between EutT and EAL nor between EutT and the EutA/EAL complex. However, growth phenotypes of a ΔeutT mutant strain show that EutT is critical for efficient ethanolamine catabolism. This work provides a preliminary understanding of the dynamics of AdoCbl synthesis and its uses within the Eut metabolosome.

Structural Basis for the Activation of the Cobalt-Carbon Bond and Control of the Adenosyl Radical in Coenzyme B12 Catalysis.

Adenosylcobalamin (AdoCbl), or coenzyme B12 , is a naturally occurring organometallic compound that serves as a cofactor for enzymes that catalyze intramolecular group-transfer reactions and ribonucleotide reduction in a wide variety of organisms from bacteria to animals. AdoCbl-dependent enzymes are radical enzymes that generate an adenosyl radical by homolysis of the coenzyme's cobalt-carbon (Co-C) bond for catalysis. How do the enzymes activate and cleave the Co-C bond to form the adenosyl radical? How do the enzymes utilize the high reactivity of the adenosyl radical for catalysis by suppressing undesirable side reactions? Our recent structural studies, which aimed to solve these problems with diol dehydratase and ethanolamine ammonia-lyase, established the crucial importance of the steric strain of the Co-C bond and conformational stabilization of the adenosyl radical for coenzyme B12 catalysis. We outline here our results obtained with these eliminating isomerases and compare them with those obtained with other radical B12 enzymes.

Metagenomic Evidence for Cobamide Producers Driving Prokaryotic Co-occurrence Associations and Potential Function in Wastewater Treatment Plants.

Cobamides are required by most organisms but are only produced by specific prokaryotic taxa. These commonly shared cofactors play significant roles in shaping the microbial community and ecosystem function. Wastewater treatment plants (WWTPs) are the world's most common biotechnological systems; knowledge about sharing of cobamides among microorganisms is predicted to be important to decipher the complex microbial relationships in these systems. Herein, we explored prokaryotic potential cobamide producers in global WWTP systems based on metagenomic analyses. A set of 8253 metagenome-assembled genomes (MAGs) were recovered and 1276 (15.5%) of them were identified as cobamide producers, which could potentially be used for the practical biological manipulation of WWTP systems. Moreover, 8090 of the total recovered MAGs (98.0%) contained at least one enzyme family dependent on cobamides, indicating the sharing of cobamides among microbial members in WWTP systems. Importantly, our results showed that the relative abundance and number of cobamide producers improved the complexity of microbial co-occurrence networks and most nitrogen, sulfur, and phosphorus cycling gene abundances, indicating the significance of cobamides in microbial ecology and their potential function in WWTP systems. These findings enhance the knowledge of cobamide producers and their functions in WWTP systems, which has important implications for improving the efficiency of microbial wastewater treatment processes.

Mobile loop dynamics in adenosyltransferase control binding and reactivity of coenzyme B12.

Cobalamin is a complex organometallic cofactor that is processed and targeted via a network of chaperones to its dependent enzymes. AdoCbl (5'-deoxyadenosylcobalamin) is synthesized from cob(II)alamin in a reductive adenosylation reaction catalyzed by adenosyltransferase (ATR), which also serves as an escort, delivering AdoCbl to methylmalonyl-CoA mutase (MCM). The mechanism by which ATR signals that its cofactor cargo is ready (AdoCbl) or not [cob(II)alamin] for transfer to MCM, is not known. In this study, we have obtained crystallographic snapshots that reveal ligand-induced ordering of the N terminus of Mycobacterium tuberculosis ATR, which organizes a dynamic cobalamin binding site and exerts exquisite control over coordination geometry, reactivity, and solvent accessibility. Cob(II)alamin binds with its dimethylbenzimidazole tail splayed into a side pocket and its corrin ring buried. The cosubstrate, ATP, enforces a four-coordinate cob(II)alamin geometry, facilitating the unfavorable reduction to cob(I)alamin. The binding mode for AdoCbl is notably different from that of cob(II)alamin, with the dimethylbenzimidazole tail tucked under the corrin ring, displacing the N terminus of ATR, which is disordered. In this solvent-exposed conformation, AdoCbl undergoes facile transfer to MCM. The importance of the tail in cofactor handover from ATR to MCM is revealed by the failure of 5'-deoxyadenosylcobinamide, lacking the tail, to transfer. In the absence of MCM, ATR induces a sacrificial cobalt-carbon bond homolysis reaction in an unusual reversal of the heterolytic chemistry that was deployed to make the same bond. The data support an important role for the dimethylbenzimidazole tail in moving the cobalamin cofactor between active sites.

Comparative Analysis of Corrinoid Profiles across Host-Associated and Environmental Samples.

Vitamin B12 (the cyanated form of cobalamin cofactors) is best known for its essential role in human health. In addition to its function in human metabolism, cobalamin also plays important roles in microbial metabolism and can impact microbial community function. Cobalamin is a member of the structurally diverse family of cofactors known as cobamides that are produced exclusively by certain prokaryotes. Cobamides are considered shared nutrients in microbial communities because the majority of bacteria that possess cobamide-dependent enzymes cannot synthesize cobamides de novo. Furthermore, different microbes have evolved metabolic specificity for particular cobamides, and therefore, the availability of cobamides in the environment is important for cobamide-dependent microbes. Determining the cobamides present in an environment of interest is essential for understanding microbial metabolic interactions. By examining the abundances of different cobamides in diverse environments, including 10 obtained in this study, we find that, contrary to its preeminence in human metabolism, cobalamin is relatively rare in many microbial habitats. Comparison of cobamide profiles of mammalian gastrointestinal samples and wood-feeding insects reveals that host-associated cobamide abundances vary and that fecal cobamide profiles differ from those of their host gastrointestinal tracts. Environmental cobamide profiles obtained from aquatic, soil, and contaminated groundwater samples reveal that the cobamide compositions of environmental samples are highly variable. As the only commercially available cobamide, cobalamin is routinely supplied during microbial culturing efforts. However, these findings suggest that cobamides specific to a given microbiome may yield greater insight into nutrient utilization and physiological processes that occur in these habitats.

Redox-Linked Coordination Chemistry Directs Vitamin B12 Trafficking.

Metals are partners for an estimated one-third of the proteome and vary in complexity from mononuclear centers to organometallic cofactors. Vitamin B12 or cobalamin represents the epitome of this complexity and is the product of an assembly line comprising some 30 enzymes. Unable to biosynthesize cobalamin, mammals rely on dietary provision of this essential cofactor, which is needed by just two enzymes, one each in the cytoplasm (methionine synthase) and the mitochondrion (methylmalonyl-CoA mutase). Brilliant clinical genetics studies on patients with inborn errors of cobalamin metabolism spanning several decades had identified at least seven genetic loci in addition to the two encoding B12 enzymes. While cells are known to house a cadre of chaperones dedicated to metal trafficking pathways that contain metal reactivity and confer targeting specificity, the seemingly supernumerary chaperones in the B12 pathway had raised obvious questions as to the rationale for their existence.With the discovery of the genes underlying cobalamin disorders, our laboratory has been at the forefront of ascribing functions to B12 chaperones and elucidating the intricate redox-linked coordination chemistry and protein-linked cofactor conformational dynamics that orchestrate the processing and translocation of cargo along the trafficking pathway. These studies have uncovered novel chemistry that exploits the innate chemical versatility of alkylcobalamins, i.e., the ability to form and dismantle the cobalt-carbon bond using homolytic or heterolytic chemistry. In addition, they have revealed the practical utility of the dimethylbenzimidazole tail, an appendage unique to cobalamins and absent in the structural cousins, porphyrin, chlorin, and corphin, as an instrument for facilitating cofactor transfer between active sites.In this Account, we navigate the chemistry of the B12 trafficking pathway from its point of entry into cells, through lysosomes, and into the cytoplasm, where incoming cobalamin derivatives with a diversity of upper ligands are denuded by the ß-ligand transferase activity of CblC to the common cob(II)alamin intermediate. The broad reaction and lax substrate specificity of CblC also enables conversion of cyanocobalamin (technically, vitamin B12, i.e., the form of the cofactor in one-a-day supplements), to cob(II)alamin. CblD then hitches up with CblC via a unique Co-sulfur bond to cob(II)alamin at a bifurcation point, leading to the cytoplasmic methylcobalamin or mitochondrial 5'-deoxyadenosylcobalamin branch. Mutations at loci upstream of the junction point typically affect both branches, leading to homocystinuria and methylmalonic aciduria, whereas mutations in downstream loci lead to one or the other disease. Elucidation of the biochemical penalties associated with individual mutations is providing molecular insights into the clinical data and, in some instances, identifying which cobalamin derivative(s) might be therapeutically beneficial.Our studies on B12 trafficking are revealing strategies for cofactor sequestration and mobilization from low- to high-affinity and low- to high-coordination-number sites, which in turn are regulated by protein dynamics that constructs ergonomic cofactor binding pockets. While these B12 lessons might be broadly relevant to other metal trafficking pathways, much remains to be learned. This Account concludes by identifying some of the major gaps and challenges that are needed to complete our understanding of B12 trafficking.

Structural Insights into the Very Low Activity of the Homocoenzyme B12 Adenosylmethylcobalamin in Coenzyme B12 -Dependent Diol Dehydratase and Ethanolamine Ammonia-Lyase.

The X-ray structures of coenzyme B12 (AdoCbl)-dependent eliminating isomerases complexed with adenosylmethylcobalamin (AdoMeCbl) have been determined. As judged from geometries, the Co-C bond in diol dehydratase (DD) is not activated even in the presence of substrate. In ethanolamine ammonia-lyase (EAL), the bond is elongated in the absence of substrate; in the presence of substrate, the complex likely exists in both pre- and post-homolysis states. The impacts of incorporating an extra CH2 group are different in the two enzymes: the DD active site is flexible, and AdoMeCbl binding causes large conformational changes that make DD unable to adopt the catalytic state, whereas the EAL active site is rigid, and AdoMeCbl binding does not induce significant conformational changes. Such flexibility and rigidity of the active sites might reflect the tightness of adenine binding. The structures provide good insights into the basis of the very low activity of AdoMeCbl in these enzymes.

Crystal structure of an atypical cobalamin riboswitch reveals RNA structural adaptability as basis for promiscuous ligand binding.

Cobalamin riboswitches encompass a structurally diverse group of cis-acting, gene regulatory elements found mostly in bacterial messenger RNA and are classified into subtypes based on secondary and tertiary characteristics. An unusual variant of the cobalamin riboswitch with predicted structural features was identified in Bacillus subtilis over a decade ago, but its structure and mechanisms of cobalamin selectivity and translational control have remained unsolved. We present the crystal structure of the aptamer domain of this atypical cobalamin riboswitch and a model for the complete riboswitch, including its expression platform domain. We demonstrate that this riboswitch binds to multiple cobalamin derivatives and correlate its promiscuous behavior to its structure and unique arrangement of peripheral elements. Comparative structural analyses between conventional cobalamin riboswitches and the B. subtilis cobalamin riboswitch reveal that the likely basis for this promiscuous ligand binding is intrinsic structural adaptability encoded in the RNA structure. It suggests that cobalamin selectivity might ultimately be viewed as existing on a spectrum of affinity for each derivative rather than as belonging to distinct types based on ligand specificities. Our work provides an interesting and notable example of functional coupling of ligand-sensing and adaptive folding by a structured RNA molecule.

Structure-Based Demystification of Radical Catalysis by a Coenzyme B12 Dependent Enzyme-Crystallographic Study of Glutamate Mutase with Cofactor Homologues.

Catalysis by radical enzymes dependent on coenzyme B12 (AdoCbl) relies on the reactive primary 5'-deoxy-5'adenosyl radical, which originates from reversible Co-C bond homolysis of AdoCbl. This bond homolysis is accelerated roughly 1012 -fold upon binding the enzyme substrate. The structural basis for this activation is still strikingly enigmatic. As revealed here, a displaced firm adenosine binding cavity in substrate-loaded glutamate mutase (GM) causes a structural misfit for intact AdoCbl that is relieved by the homolytic Co-C bond cleavage. Strategically interacting adjacent adenosine- and substrate-binding protein cavities provide a tight caged radical reaction space, controlling the entire radical path. The GM active site is perfectly structured for promoting radical catalysis, including "negative catalysis", a paradigm for AdoCbl-dependent mutases.

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.

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.

Specificity of cobamide remodeling, uptake and utilization in Vibrio cholerae.

Cobamides are a group of compounds including vitamin B12 that can vary at the lower base position of the nucleotide loop. They are synthesized de novo by only a subset of prokaryotes, but some organisms encode partial biosynthesis pathways for converting one variant to another (remodeling) or completing biosynthesis from an intermediate (corrinoid salvaging). Here, we explore the cobamide specificity in Vibrio cholerae through examination of three natural variants representing major cobamide groups: commercially available cobalamin, and isolated pseudocobalamin and p-cresolylcobamide. We show that BtuB, the outer membrane corrinoid transporter, mediates the uptake of all three variants and the intermediate cobinamide. Our previous work suggested that V. cholerae could convert pseudocobalamin produced by cyanobacteria into cobalamin. In this work, cobamide specificity in V. cholerae is demonstrated by remodeling of pseudocobalamin and salvaging of cobinamide to produce cobalamin. Cobamide remodeling in V. cholerae is distinct from the canonical pathway requiring amidohydrolase CbiZ, and heterologous expression of V. cholerae CobS was sufficient for remodeling. Furthermore, function of V. cholerae cobamide-dependent methionine synthase MetH was robustly supported by cobalamin and p-cresolylcobamide, but not pseudocobalamin. Notably, the inability of V. cholerae to produce and utilize pseudocobalamin contrasts with enteric bacteria like Salmonella.

Glycerol as a Substrate and Inactivator of Coenzyme B12 -Dependent Diol Dehydratase.

Diol dehydratase, dependent on coenzyme B12 (B12 -dDDH), displays a peculiar feature of being inactivated by its native substrate glycerol (GOL). Surprisingly, the isofunctional enzyme, B12 -independent glycerol dehydratase (B12 -iGDH), does not undergo suicide inactivation by GOL. Herein we present a series of QM/MM and MD calculations aimed at understanding the mechanisms of substrate-induced suicide inactivation in B12 -dDDH and that of resistance of B12 -iGDH to inactivation. We show that the first step in the enzymatic transformation of GOL, hydrogen abstraction, can occur from both ends of the substrate (either C1 or C3 of GOL). Whereas C1 abstraction in both enzymes leads to product formation, C3 abstraction in B12 -dDDH results in the formation of a low energy radical intermediate, which is effectively trapped within a deep well on the potential energy surface. The long lifetime of this radical intermediate likely enables its side reactions, leading to inactivation. In B12 -iGDH, by comparison, C3 abstraction is an endothermic step; consequently, the resultant radical intermediate is not of low energy, and the reverse process of reforming the reactant is possible.