Low methylcobalamin in liver tissues is an artifact as shown by a revised extraction procedure

BackgroundVitamin B12 (cobalamin, Cbl) is represented by several molecular variants distinguished by the exchangeable ligand X coordinated to cobalt ion (XCbl). The most typical XCbl-forms are cyanocobalamin (CNCbl), hydroxocobalamin (HOCbl), methylcobalamin (MeCbl) and 5′-deoxydeoxyadenosylcobalamin (AdoCbl). Cells convert the “inactive” vitamins CNCbl and HOCbl to the two critically important coenzymes AdoCbl or MeCbl. Surprisingly, little or no MeCbl is usually uncovered in the tissue samples, as compared to AdoCbl and HOCbl. We hypothesized that a low level of MeCbl is an artifact of “harsh” extractions, leading to degradation of MeCbl and/or its conversion to other XCbl-forms.MethodsWe designed a “mild” extraction protocol, including homogenization of rat liver in ammonium acetate (pH 4.6), dilution with EtOH (final 60%) and heating for 10 min at 70 °C. The XCbls were separated by HPLC and quantified by isotope dilution assays.ResultsA “mild” extraction revealed the following composition of Cbls: 37% AdoCbl, 35% MeCbl, 15% HOCbl and 13% CNCbl. The usual “harsh” protocol (pH 7, 20 min at 80 °C) changed this balance to 33%, 5%, 43% and 17%, respectively. A model assay revealed that MeCbl underwent demethylation and conversion to HOCbl at pH 3 and pH > 7, when heated with thiols. Other changes included decyanation of CNCbl and destruction of HOCbl.ConclusionsOur procedure reveals a high content of MeCbl in rat liver.General significanceThis result challenges previous data and pinpoints the need for new studies to characterize the endogenous Cbl-forms in health and disease.     

Pathogenic Mutations Differentially Affect the Catalytic Activities of the Human B12-processing Chaperone CblC and Increase Futile Redox Cycling

Human CblC catalyzes the elimination of the upper axial ligand in cobalamin or B₁₂ derivatives entering the cell from circulation. This processing step is critical for assimilation of dietary cobalamin into the active cofactor forms that support the B₁₂-dependent enzymes, methionine synthase and methylmalonyl-CoA mutase. Using a modified nitroreductase scaffold tailored to bind cobalamin and glutathione, CblC exhibits versatility in the mechanism by which it removes cyano versus alkyl ligands in cobalamin. In this study, we have characterized the effects of two pathogenic missense mutations at the same residue, R161G and R161Q, which are associated with early and late onset of the CblC disorder, respectively. We find that the R161Q and R161G CblC mutants display lower protein stability and decreased dealkylation but not decyanation activity, suggesting that cyanocobalamin might be therapeutically useful for patients carrying mutations at Arg-161. The mutant proteins also exhibit impaired glutathione binding. In the presence of physiologically relevant glutathione concentrations, stabilization of the cob(II)alamin derivative is observed, which occurs at the expense of increased oxidation of glutathione. Futile redox cycling, which is suppressed in wild-type human CblC, explains the reported increase in oxidative stress levels associated with the CblC disorder.     

Glutathione-dependent One-electron Transfer Reactions Catalyzed by a B12 Trafficking Protein

CblC is involved in an early step in cytoplasmic cobalamin processing following entry of the cofactor into the cytoplasm. CblC converts the cobalamin cargo arriving from the lysosome to a common cob(II)alamin intermediate, which can be subsequently converted to the biologically active forms. Human CblC exhibits glutathione (GSH)-dependent alkyltransferase activity and flavin-dependent reductive decyanation activity with cyanocobalamin (CNCbl). In this study, we discovered two new GSH-dependent activities associated with the Caenorhabditis elegans CblC for generating cob(II)alamin: decyanation of CNCbl and reduction of aquocobalamin (OH₂Cbl). We subsequently found that human CblC also catalyzes GSH-dependent decyanation of CNCbl and reduction of OH₂Cbl, albeit efficiently only under anaerobic conditions. The air sensitivity of the human enzyme suggests interception by oxygen during the single-electron transfer step from GSH to CNCbl. These newly discovered GSH-dependent single-electron transfer reactions expand the repertoire of catalytic activities supported by CblC, a versatile B₁₂-processing enzyme.     

Decreased Brain Levels of Vitamin B12 in Aging,Autism and Schizophrenia

Many studies indicate a crucial role for the vitamin B₁₂ and folate-dependent enzyme methionine synthase (MS) in brain development and function, but vitamin B₁₂ status in the brain across the lifespan has not been previously investigated. Vitamin B₁₂ (cobalamin, Cbl) exists in multiple forms, including methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl), serving as cofactors for MS and methylmalonylCoA mutase, respectively. We measured levels of five Cbl species in postmortem human frontal cortex of 43 control subjects, from 19 weeks of fetal development through 80 years of age, and 12 autistic and 9 schizophrenic subjects. Total Cbl was significantly lower in older control subjects (> 60 yrs of age), primarily reflecting a >10-fold age-dependent decline in the level of MeCbl. Levels of inactive cyanocobalamin (CNCbl) were remarkably higher in fetal brain samples. In both autistic and schizophrenic subjects MeCbl and AdoCbl levels were more than 3-fold lower than age-matched controls. In autistic subjects lower MeCbl was associated with decreased MS activity and elevated levels of its substrate homocysteine (HCY). Low levels of the antioxidant glutathione (GSH) have been linked to both autism and schizophrenia, and both total Cbl and MeCbl levels were decreased in glutamate-cysteine ligase modulatory subunit knockout (GCLM-KO) mice, which exhibit low GSH levels. Thus our findings reveal a previously unrecognized decrease in brain vitamin B₁₂ status across the lifespan that may reflect an adaptation to increasing antioxidant demand, while accelerated deficits due to GSH deficiency may contribute to neurodevelopmental and neuropsychiatric disorders.     

Glutathione increases the binding affinity of a bovine B12 trafficking chaperone bCblC for vitamin B12

Intracellular B₁₂ metabolism involves a B₁₂ trafficking chaperone CblC that is well conserved in mammals including human. The protein CblC is known to bind cyanocobalamin (CNCbl, vitamin B₁₂) inducing the base-off transition and convert it into an intermediate that can be used in enzyme cofactor synthesis. The binding affinity of human CblC for CNCbl was determined to be K_d = ≈6–16 μM, which is relatively low considering sub-micromolar B₁₂ concentrations (0.03–0.7 μM) in normal cells. In the current study, we discovered that the base-off transition of CNCbl upon binding to bCblC, a bovine homolog of human CblC, is facilitated in the presence of reduced form of glutathione (GSH). In addition, GSH dramatically increases the binding affinity for CNCbl lowering the K_d from 27.1 ± 0.2–0.24 ± 0.09 μM. The effect of GSH is due to conformational change of bCblC upon binding with GSH, which was indicated by limited proteolysis and urea-induced equilibrium denaturation of the protein. The results of this study suggest that GSH positively modulates bCblC by increasing the binding affinity for CNCbl, which would enhance functional efficiency of the protein.     

Structural Insights into the MMACHC-MMADHC Protein Complex Involved in Vitamin B12 Trafficking

Conversion of vitamin B₁₂ (cobalamin, Cbl) into the cofactor forms methyl-Cbl (MeCbl) and adenosyl-Cbl (AdoCbl) is required for the function of two crucial enzymes, mitochondrial methylmalonyl-CoA mutase and cytosolic methionine synthase, respectively. The intracellular proteins MMACHC and MMADHC play important roles in processing and targeting the Cbl cofactor to its destination enzymes, and recent evidence suggests that they may interact while performing these essential trafficking functions. To better understand the molecular basis of this interaction, we have mapped the crucial protein regions required, indicate that Cbl is likely processed by MMACHC prior to interaction with MMADHC, and identify patient mutations on both proteins that interfere with complex formation, via different mechanisms. We further report the crystal structure of the MMADHC C-terminal region at 2.2 Å resolution, revealing a modified nitroreductase fold with surprising homology to MMACHC despite their poor sequence conservation. Because MMADHC demonstrates no known enzymatic activity, we propose it as the first protein known to repurpose the nitroreductase fold solely for protein-protein interaction. Using small angle x-ray scattering, we reveal the MMACHC-MMADHC complex as a 1:1 heterodimer and provide a structural model of this interaction, where the interaction region overlaps with the MMACHC-Cbl binding site. Together, our findings provide novel structural evidence and mechanistic insight into an essential biological process, whereby an intracellular “trafficking chaperone” highly specific for a trace element cofactor functions via protein-protein interaction, which is disrupted by inherited disease mutations.     

Structural basis of multifunctionality in a vitamin B12-processing enzyme

An early step in the intracellular processing of vitamin B(12) involves CblC, which exhibits dual reactivity, catalyzing the reductive decyanation of cyanocobalamin (vitamin B(12)), and the dealkylation of alkylcobalamins (e.g. methylcobalamin; MeCbl). Insights into how the CblC scaffold supports this chemical dichotomy have been unavailable despite it being the most common locus of patient mutations associated with inherited cobalamin disorders that manifest in both severe homocystinuria and methylmalonic aciduria. Herein, we report structures of human CblC, with and without bound MeCbl, which provide novel biochemical insights into its mechanism of action. Our results reveal that CblC is the most divergent member of the NADPH-dependent flavin reductase family and can use FMN or FAD as a prosthetic group to catalyze reductive decyanation. Furthermore, CblC is the first example of an enzyme with glutathione transferase activity that has a sequence and structure unrelated to the GST superfamily. CblC thus represents an example of evolutionary adaptation of a common structural platform to perform diverse chemistries. The CblC structure allows us to rationalize the biochemical basis of a number of pathological mutations associated with severe clinical phenotypes.     

Towards the synthesis of light-stable coenzyme B12 analogs

The organometallic complex coenzyme B₁₂ (adenosyl cobalamin, AdoCbl) is not only an essential coenzyme in many biochemical reactions of most if not all living organisms but has lately been shown to play a crucial role in the regulation of B₁₂ related genes. As a consequence, coenzyme B₁₂ has been a target of intense research. However, the investigations of AdoCbl have often been hampered due to its high light-sensitivity leading to decomposition of the compound within a few seconds. Here, we describe a strategy to synthesize more light-stable coenzyme B₁₂ analogs, which show similar steric properties as adenosyl cobalamin. The synthesis, structural characterization as well as the pH dependent “base-on/base-off” behavior of cyanide bridged vitamin B₁₂ conjugates with either a cis-[(NH₃)₂Pt]²⁺ or an [enPt]²⁺ moiety, leading to cis-[(NH₃)₂PtCl-vitB₁₂]⁺ (1) and [enPtCl-vitB₁₂]⁺ (2) are reported. The subsequent reaction of cis-[(NH₃)₂PtCl-vitB₁₂]⁺ with the model nucleobase 9-methyladenine leads to the corresponding adduct, where the adenine moiety is coordinated to the Pt²⁺ center either via N1 or N7. This compound is light-stable and harbors the adenine moiety in the same distance of 5 Å above the corrin plane as present in the highly light-sensitive adenosyl cobalamin.     

Microbiological activities of nucleotide loop-modified analogues of vitamin B12

Novel vitamin B₁₂ analogues in which the D-ribose moiety of the nucleotide loop was replaced by an oligomethylene group and a trimethylene analogue containing imidazole instead of 5,6-dimethylbenzimidazole as well as cobinamide methyl phosphate were tested for biological activities with Escherichia coli 215, a B₁₂- or methionine-auxotroph, and Lactobacillus leichmannii ATCC 7830 as test organisms. A cyano form of 5,6-dimethylbenzimidazolyl tetramethylene, trimethylene and hexamethylene analogues supported the growth of L. leichmannii in this order. 5.6-Dimethylbenzimidazolyl dimethylene and imidazolyl trimethylene analogues did not show B₁₂ activity and behaved as weak B₁₂ antagonists when added together with cyanocobalamin. An adenosyl form of the biologically active analogues served as coenzymes for ribonucleotide reductase of this bacterium, whereas that of the inactive analogues did not. The latter acted as weak competitive inhibitors against adenosylcobalamin. ON the contrary, all the analogues did not support the growth of E. coli 215 at all by themselves and inhibited the growth when added with a suboptimum level of cyanocobalamin. A methyl form of the analogues also did not support the growth of E. coli 215, although they served as active coenzymes for methionine synthase of the bacterium. Since unlabeled analogues strongly inhibited the uptake of [³H]cyanocobalamin by this bacterium, it seems likely that the analogues exert their anti-B₁₂ activity toward E. coli 215 by blocking the B₁₂-transport systemAbbreviations AdoCbl adenosylcobalamin - MeCbl methylcobalamin - CN-Cbl cyanocobalamin or vitamin B₁₂ - Cbl cobalamin - (CN, aq)Cbi cyanoaquacobinamide - MeCbi methylcobinamide - Cbi cobinamide - (CN, aq)Cbi-PMe cyanoaquacobinamide methyl phosphate - Cbi-PMe cobinamide methyl phosphate - DBI 5,6-dimethylbenzimidazole - DBIyl 5,6-dimethylbenzimidazolyl - FMNH₂ fully reduced form of riboflavin 5-phosphate     

The mechanism of action of ethanolamine ammonia-lyase, an adenosylcobalamin-dependent enzyme. Reaction of the enzyme.cofactor complex with 2-aminoacetaldehyde

Ethanolamine ammonia-lyase (EC 4.3.1.7) catalyzes the adenosylcobalamin-dependent deamination of ethanolamine and 2-aminopropanol. Incubation of the enzyme.cofactor complex with 2-aminoacetaldehyde leads to rapid cleavage of the carbon--cobalt bond accompanied by the destruction of the corrinoid portion of the cofactor. During this reaction the adenosyl portion of the cofactor is oxidized to 4',5'-anhydroadenosine, and the aminoacetaldehyde is converted to acetic acid, which remains associated with the enzyme as a noncovalent complex which survives gel filtration. There is no evidence for the alkylation of the corrin metal by the substrate analog. The enzyme.AdoCbl complex is thus able to eliminate an amino group from a substrate analog without the formation of a new alkyl cobalamin in which the analog is a ligand. These observations do not support the participation of what might be termed "substratylcobalamin" as an intermediate in the ammonia migration occurring in reactions catalyzed by ethanolamine ammonia-lyase.     

Assembly and protection of the radical enzyme, methylmalonyl-CoA mutase, by its chaperone

MeaB is a recently described P-loop GTPase that plays an auxiliary role in the reaction catalyzed by the radical B12 enzyme, methylmalonyl-CoA mutase. Defects in the human homologue of MeaB result in methylmalonic aciduria, but the role of this protein in coenzyme B12 assimilation and/or utilization is not known. Methylmalonyl-CoA mutase catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA that uses reactive radical intermediates that are susceptible to oxidative inactivation. In this study, we have examined the influence of MeaB on the kinetics of the reaction catalyzed by methylmalonyl-CoA mutase and on the thermodynamics of cofactor binding. MeaB alone has a modest effect on the affinity of the mutase for the 5'-deoxyadenosylcobalamin (AdoCbl) cofactor, increasing it 2-fold from 404 +/- 71 to 210 +/- 22 nM. However, in the presence of GDP, the affinity for the cofactor decreases 5-fold to 1.89 +/- 0.33 microM, while in the presence of guanosine 5'(beta-gamma imino)triphosphate, a nonhydrolyzable analogue of GTP, the binding of AdoCbl to the mutase is not detected. Protection against oxidative inactivation of the mutase by MeaB is dependent upon the presence of nucleotides with the MeaB/GDP and MeaB/GTP complexes decelerating the rate of formation of oxidized cofactor by 3- and 15-fold, respectively. This study suggests that MeaB functions in the GTP-dependent assembly of holomethylmalonyl-CoA mutase and subsequent protection of radical intermediates during catalysis.     

The Aeration-Dependent Effect of Vitamin B12 on DNA Biosynthesis in Methylobacterium dichloromethanicum

The effect of vitamin B₁₂ (cobalamin) on DNA biosynthesis in Methylobacterium dichloromethanicum was studied. When cultivated in media with methanol or dichloromethane, the bacterium produced approximately 10 g corrinoids per gram dry biomass, compared to about 7 g/g when cultivated on ethanol or succinate. Exogenous adenosylcobalamin (AdoCbl) stimulated DNA biosynthesis in M. dichloromethanicum cells grown under poor aeration, the effect being mediated by AdoCbl-dependent ribonucleotide reductase. In vitro studies showed that M. dichloromethanicumalso has AdoCbl-independent ribonucleotide reductase. Under good aeration, exogenous AdoCbl had no effect on DNA biosynthesis, while hydroxyurea suppressed it. These data suggest that AdoCbl-independent ribonucleotide reductase, which is likely to be activated by oxygen, plays an important part in DNA biosynthesis when M. dichloromethanicum is cultured with good aeration, whereas AdoCbl-dependent ribonucleotide reductase is active under conditions of poor aeration.     

Patient mutations in human ATP:cob(I)alamin adenosyltransferase differentially affect its catalytic versus chaperone functions

Human ATP:cob(I)alamin adenosyltransferase (ATR) is a mitochondrial enzyme that catalyzes an adenosyl transfer to cob(I)alamin, synthesizing 5′-deoxyadenosylcobalamin (AdoCbl) or coenzyme B₁₂. ATR is also a chaperone that escorts AdoCbl, transferring it to methylmalonyl-CoA mutase, which is important in propionate metabolism. Mutations in ATR lead to methylmalonic aciduria type B, an inborn error of B₁₂ metabolism. Our previous studies have furnished insights into how ATR protein dynamics influence redox-linked cobalt coordination chemistry, controlling its catalytic versus chaperone functions. In this study, we have characterized three patient mutations at two conserved active site residues in human ATR, R190C/H, and E193K and obtained crystal structures of R190C and E193K variants, which display only subtle structural changes. All three mutations were found to weaken affinities for the cob(II)alamin substrate and the AdoCbl product and increase K_M(ATP). ³¹P NMR studies show that binding of the triphosphate product, formed during the adenosylation reaction, is also weakened. However, although the k_cat of this reaction is significantly diminished for the R190C/H mutants, it is comparable with the WT enzyme for the E193K variant, revealing the catalytic importance of Arg-190. Furthermore, although the E193K mutation selectively impairs the chaperone function by promoting product release into solution, its catalytic function might be unaffected at physiological ATP concentrations. In contrast, the R190C/H mutations affect both the catalytic and chaperoning activities of ATR. Because the E193K mutation spares the catalytic activity of ATR, our data suggest that the patients carrying this mutation are more likely to be responsive to cobalamin therapy.     

Purification and interaction analyses of two human lysosomal vitamin B12 transporters: LMBD1 and ABCD4

Mutations in human LMBRD1 and ABCD4 prevent lysosomal export of vitamin B₁₂ to the cytoplasm, impairing the vitamin B₁₂-dependent enzymes methionine synthase and methylmalonyl-CoA mutase. The gene products of LMBRD1 and ABCD4 are implicated in vitamin B₁₂ transport at the lysosomal membrane and are proposed to act in complex. To address the mechanism for lysosomal vitamin B₁₂ transport, we report the novel recombinant production of LMBD1 and ABCD4 for detailed biophysical analyses. Using blue native PAGE, chemical crosslinking, and size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), we show that both detergent-solubilized LMBD1 and detergent-solubilized ABCD4 form homodimers. To examine the functional binding properties of these proteins, label-free surface plasmon resonance (SPR) provides direct in vitro evidence that: (i) LMBD1 and ABCD4 interact with low nanomolar affinity; and (ii) the cytoplasmic vitamin B₁₂-processing protein MMACHC also interacts with LMBD1 and ABCD4 with low nanomolar affinity. Accordingly, we propose a model whereby membrane-bound LMBD1 and ABCD4 facilitate the vectorial delivery of lysosomal vitamin B₁₂ to cytoplasmic MMACHC, thus preventing cofactor dilution to the cytoplasmic milieu and protecting against inactivating side reactions.     

C-terminal truncation of a bovine B12 trafficking chaperone enhances the sensitivity of the glutathione-regulated thermostability

The human B₁₂ trafficking chaperone hCblC is well conserved in mammals and non-mammalian eukaryotes. However, the C-terminal ∼40 amino acids of hCblC vary significantly and are predicted to be deleted by alternative splicing of the encoding gene. In this study, we examined the thermostability of the bovine CblC truncated at the C-terminal variable region (t-bCblC) and its regulation by glutathione. t-bCblC is highly thermolabile (T_m = ∼42℃) similar to the full-length protein (f-bCblC). However, t-bCblC is stabilized to a greater extent than f-bCblC by binding of reduced glutathione (GSH) with increased sensitivity to GSH. In addition, binding of oxidized glutathione (GSSG) destabilizes t-bCblC to a greater extent and with increased sensitivity as compared to f-bCblC. These results indicate that t-bCblC is a more sensitive form to be regulated by glutathione than the full-length form of the protein. [BMB Reports 2013; 46(3): 169-174]     

The cobinamide amidohydrolase (cobyric acid‐forming) CbiZ enzyme: a critical activity of the cobamide remodelling system of Rhodobacter sphaeroides

The chemical structures of cobamides [cobalamin (Cbl)‐like compounds] are the same, except for the lower ligand, which in adenosylcobalamin (AdoCbl) is 5,6‐dimethylbenzimidazole, and in adenosylpseudocobalamin (AdopseudoCbl) is adenine. Why the lower ligand of cobamides varies and what the mechanism of lower ligand replacement is are long‐standing questions in the field of B₁₂ biosynthesis. Work reported here uncovers the strategy used by the photosynthetic α‐proteobacterium Rhodobacter sphaeroides to procure the cobamide it needs to grow on acetate as a carbon and energy source. On the basis of genetic and biochemical evidence we conclude that, in R. sphaeroides, the activity of the cobyric acid‐producing amidohydrolase CbiZ enzyme is essential for the conversion of AdopseudoCbl into AdoCbl, the cobamide needed for the catabolism of acetate. The CbiZ enzyme uses AdopseudoCbl as a substrate, but not AdoCbl. Implications of these findings for cobamide remodelling in R. sphaeroides and in other CbiZ‐containing microorganisms are discussed.     

Interactions of methylmalonyl CoA mutase from normal human fibroblasts with adenosylcobalamin and methylmalonyl CoA: evidence for non-equivalent active sites

We have examined interactions between human methylmalonyl CoA mutase and two critical ligands, its cofactor adenosylcobalamin (AdoCbl) and its substrate methylmalonyl CoA, by performing in vitro experiments with preparations of mutase apoenzyme and holoenzyme from normal cultured human fibroblasts. When extracts are prepared from cells grown in medium containing high concentrations of hydroxocobalamin, a precursor of AdoCbl, mutase activity measured in Tris-containing buffers in the absence of added AdoCbl accounts maximally for only 50% of that activity measured in the presence of excess AdoCbl. A similar result is observed when mutase holoenzyme is formed in vitro by incubating cell extracts containing apoenzyme with AdoCbl and removing excess AdoCbl by gel filtration. When such holoenzyme preparations are heated at 45 °C and then assayed for activity, their thermostability is less than that of mutase holoenzyme heated in the presence of excess cofactor, but far greater than that of mutase apoenzyme. Methylmalonyl CoA modulates these enzyme-coenzyme interactions, since mutase holoenzyme formed in Triscontaining buffers is resolved to apoenzyme upon exposure to substrate. Qualitatively different data are obtained when buffers containing cations other than Tris are used. Under these conditions, mutase activity measured in the absence of added AdoCbl accounts for nearly 100% of the activity measured in the presence of excess cofactor, whether holoenzyme is formed in intact cells in culture or in cell extracts in vitro. Furthermore, holoenzyme formed in vitro in potassium phosphate buffer is not resolved to apoenzyme upon exposure to substrate. We suggest that the “holoenzyme” form of mutase obtained and assayed in Tris-containing buffers is that molecular species with only one of its two potential AdoCbl binding sites occupied in a catalytically active fashion, and that other ions can influence markedly the interactions between mutase, AdoCbl, and methylmalonyl CoA. These data are consistent, therefore, with the hypothesis that the dimeric mutase apoenzyme is characterized, under certain conditions, by nonequivalent active sites.     

Aquatic metagenomes implicate Thaumarchaeota in global cobalamin production

Cobalamin (vitamin B₁₂) is a complex metabolite and essential cofactor required by many branches of life, including most eukaryotic phytoplankton. Algae and other cobalamin auxotrophs rely on environmental cobalamin supplied from a relatively small set of cobalamin-producing prokaryotic taxa. Although several Bacteria have been implicated in cobalamin biosynthesis and associated with algal symbiosis, the involvement of Archaea in cobalamin production is poorly understood, especially with respect to the Thaumarchaeota. Based on the detection of cobalamin synthesis genes in available thaumarchaeotal genomes, we hypothesized that Thaumarchaeota, which are ubiquitous and abundant in aquatic environments, have an important role in cobalamin biosynthesis within global aquatic ecosystems. To test this hypothesis, we examined cobalamin synthesis genes across sequenced thaumarchaeotal genomes and 430 metagenomes from a diverse range of marine, freshwater and hypersaline environments. Our analysis demonstrates that all available thaumarchaeotal genomes possess cobalamin synthesis genes, predominantly from the anaerobic pathway, suggesting widespread genetic capacity for cobalamin synthesis. Furthermore, although bacterial cobalamin genes dominated most surface marine metagenomes, thaumarchaeotal cobalamin genes dominated metagenomes from polar marine environments, increased with depth in marine water columns, and displayed seasonality, with increased winter abundance observed in time-series datasets (e.g., L4 surface water in the English Channel). Our results also suggest niche partitioning between thaumarchaeotal and cyanobacterial ribosomal and cobalamin synthesis genes across all metagenomic datasets analyzed. These results provide strong evidence for specific biogeographical distributions of thaumarchaeotal cobalamin genes, expanding our understanding of the global biogeochemical roles played by Thaumarchaeota in aquatic environments.     

Riboswitch effectors as protein enzyme cofactors

The recently identified glmS ribozyme revealed that RNA enzymes, like protein enzymes, are capable of using small molecules as catalytic cofactors to promote chemical reactions. Flavin mononucleotide (FMN), S-adenosyl methionine (SAM), adenosyl cobalamin (AdoCbl), and thiamine pyrophosphate (TPP) are known ligands for RNA riboswitches in the control of gene expression, but are also catalytically powerful and ubiquitous cofactors in protein enzymes. If RNA, instead of just binding these molecules, could harness the chemical potential of the cofactor, it would significantly expand the enzymatic repertoire of ribozymes. Here we review the chemistry of AdoCbl, SAM, FMN, and TPP in protein enzymology and speculate on how these cofactors might have been used by ribozymes in the prebiotic RNA World or may still find application in modern biology.     

Mutualistic interactions between vitamin B(12) -dependent algae and heterotrophic bacteria exhibit regulation

Many algae are auxotrophs for vitamin B₁₂ (cobalamin), which they need as a cofactor for B₁₂‐dependent methionine synthase (METH). Because only prokaryotes can synthesize the cobalamin, they must be the ultimate source of the vitamin. In the laboratory, a direct interaction between algae and heterotrophic bacteria has been shown, with bacteria supplying cobalamin in exchange for fixed carbon. Here we establish a system to study this interaction at the molecular level. In a culture of a B₁₂‐dependent green alga Chlamydomonas nivalis, we found a contaminating bacterium, identified by 16S rRNA analysis as Mesorhizobium sp. Using the sequenced strain of M. loti (MAFF303099), we found that it was able to support the growth of B₁₂‐dependent Lobomonas rostrata, another green alga, in return for fixed carbon. The two organisms form a stable equilibrium in terms of population numbers, which is maintained over many generations in semi‐continuous culture, indicating a degree of regulation. However, addition of either vitamin B₁₂ or a carbon source for the bacteria perturbs the equilibrium, demonstrating that the symbiosis is mutualistic and facultative. Chlamydomonas reinhardtii does not require B₁₂ for growth because it encodes a B₁₂‐independent methionine synthase, METE, the gene for which is suppressed by addition of exogenous B₁₂. Co‐culturing C. reinhardtii with M. loti also results in reduction of METE expression, demonstrating that the bacterium can deliver the vitamin to this B₁₂‐independent alga. We discuss the implications of this for the widespread distribution of cobalamin auxotrophy in the algal kingdom.