Fluorinated vitamin b(12) analogs are cofactors of corrinoid-dependent enzymes: a f-labeled nuclear magnetic resonance probe for identifying corrinoid-protein interactions

The homoacetogenic bacterium Sporomusa ovata synthesized the vitamin B(12) analog phenolyl cobamide or 4-fluorophenolyl cobamide when the methanol medium of growing cells was supplemented with 10 mM phenol or 5 mM 4-fluorophenol. Phenol and, presumably, 4-fluorophenol were specifically incorporated into these cobamides, since phenol was not metabolized significantly into amino acids or into acetic acid, the product of the catabolism. The phenol-containing cobamides contributed up to 90% of the protein-bound cobamides of the 1,300 to 1,900 nmol of corrinoid per g of dry cell material formed. Fluorine-19 nuclear magnetic resonance spectroscopy of 4-fluorophenolyl cobamide exhibited a resonance near 30 ppm. An additional signal emerged at 25 ppm when 4-fluorophenolyl cobamide was investigated as the cofactor of a corrinoid-dependent protein. The two resonances indicated distinct cofactor arrangements within the protein's active site. A 5-ppm high-field shift change suggested van der Waal's interactions between the fluorinated nucleotide of the cofactor and adjacent amino acid residues of the enzyme. Similarly, Propionibacterium freudenreichii and Methanobacterium thermoautotrophicum synthesized 5-fluorobenzimidazolyl cobamide. The human corrinoid binders intrinsic factor, transcobalamin, and haptocorrin recognized this corrinoid like vitamin B(12). Hence, it is possible to use F-labeled nuclear magnetic resonance spectroscopy for analyses of protein-bound cobamides.     

Identification of phenolyl cobamide from the homoacetogenic bacterium Sporomusa ovata

Phenolyl cobamide was isolated from cyanide extractions of the anaerobic eubacterium Sporomusa ovata. The proposed corrinoid structure [Co alpha,Co beta-(monocyano,monoaquo)-phenolyl cobamide] has been deduced from 1H NMR, fast-atom-bombardment mass spectroscopy and ultraviolet/visible spectroscopy data. The complete corrinoid resembled p-cresolyl cobamide [Co alpha,Co beta-(monocyano,monoaquo)-p-cresolyl cobamide], which recently has been obtained from cyanide extractions of the same bacterium. The structures and chemical properties of both cobamides with uncoordinated nucleotides differed significantly from those of vitamin B12 [Co alpha-[alpha-(5,6-dimethylbenzimidazolyl)]-Co beta-cyanocobamide]. Sporomusa synthesized coenzymes of phenolyl cobamide and p-cresolyl cobamide in considerable amounts of 400 nmol/g and 1700 nmol/g dry cells, respectively. More than 90% of the complete corrinoid pool of the homoacetogenic bacterium consisted of these two corrinoids, indicating that they are physiologically important coenzymes of the bacterial metabolism.     

Isolation of cobamides from Methanothrix soehngenii: 5-methylbenzimidazole as the α-ligand of the predominant cobamide

Methanothrix soehngenii was found to contain five different cobamides when grown on vitamin B₁₂ supplemented as well as vitamin B₁₂ free media. In both cases, it was shown by HPLC-chromatography and UV/VIS spectroscopy, that -5-methylbenzimidazolyl--cyanocobamide was the predominant cobamide, accounting for 27% and 23%, respectively, of the total corrinoid content. Vitamin B₁₂ and -5-hydroxybenzimidazolyl--cyanocobamide could also be detected in both cell batches in varying amounts. Cells grown on vitamin B₁₂ free medium contained significantly more baseless cobamides, indicating biosynthesis of cobamides.Abbreviations (5-MeBza)CNCba -5-methylbenzimidazolyl--cyanocobamide - (5-HOBza)CNCBa -5-hydroxybenzimidazolyl--cyanocobamide (factor III) - (5-MeOBza)CNCba -5-methoxybenzimidazolyl--cyanocobamide (factor III_m) - (Bza)CNCba -benzimidazolyl--cyanocobamide     

Characterization of vitamin B12 compounds in the fruiting bodies of shiitake mushroom (Lentinula edodes) and bed logs after fruiting of the mushroom

This study determined the vitamin B₁₂ content in commercially available dried fruiting bodies of shiitake mushroom, Lentinula edodes. The vitamin B₁₂ contents in dried donko-type fruiting bodies with closed caps (5.61 ± 3.90 μg/100 g dry weight), did not significantly differ from those of dried koushin-type fruiting bodies with open caps (4.23 ± 2.42 μg/100 g dry weight). The bed logs after fruiting of the mushroom also contained the vitamin B₁₂ levels similar to that in the dried shiitake fruiting bodies. To determine whether the dried shiitake fruiting bodies and their bed logs contained vitamin B₁₂ or other corrinoid compounds that are inactive in humans, we purified corrinoid compounds using an immunoaffinity column and identified vitamin B₁₂ using vitamin B₁₂-dependent Escherichia coli 215 bioautograms and liquid chromatography-electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) chromatograms. Dried shiitake fruiting bodies rarely contained an unnatural corrinoid vitamin B₁₂[c-lactone] that is inactive in humans. Given that shiitake mushroom lacks the ability to synthesize vitamin B₁₂ de novo, the vitamin B₁₂ found in dried shiitake fruiting bodies must have been derived from the bed logs.     

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.     

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.     

Characterization of a Corrinoid Compound in the Edible (Blue-Green) Alga,Suizenji-nori

The edible blue-green alga (cyanobacterium), Suizenji-nori, contained 143.8±22.4 μg of vitamin B₁₂ per 100 g dry weight of the alga (mean±SE, n=4). A corrinoid compound was purified from the dried Suizenji-nori, and partially characterized. The silica gel 60 TLC and reversed-phase HPLC patterns of the purified corrinoid compound were not identical to those of true vitamin B₁₂, but to those of pseudovitamin B₁₂ which is inactive for humans.     

Impact of Vitamin B12 on Formation of the Tetrachloroethene Reductive Dehalogenase in Desulfitobacterium hafniense Strain Y51

Corrinoids are essential cofactors of reductive dehalogenases in anaerobic bacteria. Microorganisms mediating reductive dechlorination as part of their energy metabolism are either capable of de novo corrinoid biosynthesis (e.g., Desulfitobacterium spp.) or dependent on exogenous vitamin B₁₂ (e.g., Dehalococcoides spp.). In this study, the impact of exogenous vitamin B₁₂ (cyanocobalamin) and of tetrachloroethene (PCE) on the synthesis and the subcellular localization of the reductive PCE dehalogenase was investigated in the Gram-positive Desulfitobacterium hafniense strain Y51, a bacterium able to synthesize corrinoids de novo. PCE-depleted cells grown for several subcultivation steps on fumarate as an alternative electron acceptor lost the tetrachloroethene-reductive dehalogenase (PceA) activity by the transposition of the pce gene cluster. In the absence of vitamin B₁₂, a gradual decrease of the PceA activity and protein amount was observed; after 5 subcultivation steps with 10% inoculum, more than 90% of the enzyme activity and of the PceA protein was lost. In the presence of vitamin B₁₂, a significant delay in the decrease of the PceA activity with an ∼90% loss after 20 subcultivation steps was observed. This corresponded to the decrease in the pceA gene level, indicating that exogenous vitamin B₁₂ hampered the transposition of the pce gene cluster. In the absence or presence of exogenous vitamin B₁₂, the intracellular corrinoid level decreased in fumarate-grown cells and the PceA precursor formed catalytically inactive, corrinoid-free multiprotein aggregates. The data indicate that exogenous vitamin B₁₂ is not incorporated into the PceA precursor, even though it affects the transposition of the pce gene cluster.     

Substitution of Co alpha-(5-hydroxybenzimidazolyl)cobamide (factor III) by vitamin B12 in Methanobacterium thermoautotrophicum.

Methanobacterium thermoautotrophicum grown on mineral medium contains 120 nmol of Co alpha-(5-hydroxybenzimidazolyl)cobamides (derivatives of factor III) per g of dry cell mass as the sole cobamide. The bacterium assimilated several corrinoids and benzimidazole bases during autotrophic growth. The corrinoids were converted into factor III; however, after three transfers in 5,6-dimethylbenzimidazole (200 microM)-supplemented mineral medium, derivatives of factor III were completely replaced by derivatives of vitamin B12, which is atypical for methanogens. The total cobamide content of these cells and their growth rate were not affected compared with factor III-containing cells. Therefore, the high cobamide content rather than a particular type of cobamide is required for metabolism of methanogens. Derivatives of factor III are not essential cofactors of cobamide-containing enzymes from methanogenic bacteria, but they are the result of a unique biosynthetic ability of these archaebacteria. The cobamide biosynthesis include unspecific enzymes, which made it possible either to convert non-species-derived corrinoids into derivatives of factor III or to synthesize other types of cobamides than factor III. The cobamide biosynthesis is regulated by its end product. In addition, the uptake of extracellular cobamides is controlled, and the assimilated corrinoids regulate cellular cobamide biosynthesis.     

Identification of Fluoropyrogallols as New Intermediates in Biotransformation of Monofluorophenols in Rhodococcus opacus 1cp

The transformation of monofluorophenols by whole cells of Rhodococcus opacus 1cp was investigated, with special emphasis on the nature of hydroxylated intermediates formed. Thin-layer chromatography, mass spectrum analysis, and ¹⁹F nuclear magnetic resonance demonstrated the formation of fluorocatechol and trihydroxyfluorobenzene derivatives from each of three monofluorophenols. The ¹⁹F chemical shifts and proton-coupled splitting patterns of the fluorine resonances of the trihydroxyfluorobenzene products established that the trihydroxylated aromatic metabolites contained hydroxyl substituents on three adjacent carbon atoms. Thus, formation of 1,2,3-trihydroxy-4-fluorobenzene (4-fluoropyrogallol) from 2-fluorophenol and formation of 1,2,3-trihydroxy-5-fluorobenzene (5-fluoropyrogallol) from 3-fluorophenol and 4-fluorophenol were observed. These results indicate the involvement of fluoropyrogallols as previously unidentified metabolites in the biotransformation of monofluorophenols in R. opacus 1cp.     

Pseudo vitamin B12 or 5-hydroxybenzimidazolyl-cobamide are the corrinoids found in methanogenic bacteria

Thirteen species of methanogenic bacteria were analyzed for corrinoids. Pseudo vitamin B₁₂ (Co-[-(7-adenyl)]-cobamide) was the predominant cobamide of methanococcales and Methanoplanus. All other methanogens contained factor III (Co-[-(5-hydroxybenzimidazolyl)]-cobamide). Vitamin B₁₂ (Co-[-(5,6-dimethylbenzimidazolyl)]-cobamide) was not detected in any of these archaebacteria. Their cobamide content was 100 to 1400 nmol per gram cell dry weight, indicating that abundant cobamides are essential for methanogens.     

Anaerobic degradation of tetrachloromethane by Acetobacterium woodii: separation of dechlorinative activities in cell extracts and roles for vitamin B12 and other factors

The transformations of ¹⁴CCl₄ by whole cells of Acetobacterium woodii suspended in phosphate buffer containing reducing agents, and by the cobalt corrinoid aquocobalamin in the same solution, were compared. Each catalyst transformed ¹⁴CCl₄ not only to reduced products (CHCl₃ and CH₂Cl₂) but also to CO and CO₂ as well as non-volatile products. The mass balance for radioactive carbon was complete in each case. Thus, the reactions of the pure cobalt corrinoid resemble the reactions in vivo. The proton in CHCl₃, formed from CCl₄ by A. woodii, was derived from water. Extracts of A. woodii were fractionated into large and small molecules, and each of the two fractions was separated chromatographically. Fractions of proteins demonstrated poor correlation between content of the corrinoid vitamin B₁₂ and rates of transformation of CCl₄. The correlation was somewhat improved if the fractions were autoclaved, but dechlorination in the absence of vitamin B₁₂ was observed. Separation of the small molecules yielded only one fraction containing vitamin B₁₂, and this fraction catalyzed dechlorination, whereas several other fractions were able to dechlorinate CCl₄ in the absence of vitamin B₁₂. We presume there to be unrecognized dechlorinative factors in anaerobic bacteria.Abbreviations GC gas chromatograph(y) - GC-MS (or-TCD or-FID) GC coupled to a mass spectrometer (or a thermal conductivity detector or a flame ionization detector) - HPLC high pressure liquid chromatograph(y) - FPLC high pressure protein chromatograph(y)     

Spectroelectrochemical studies of the corrinoid/iron-sulfur protein involved in acetyl coenzyme A synthesis by Clostridium thermoaceticum

An 88-kDa corrinoid/iron-sulfur protein (C/Fe-SP) is the methyl carrier protein in the acetyl-CoA pathway of Clostridium thermoaceticum. In previous studies, it was found that this C/Fe-SP contains (5-methoxybenzimidazolyl)cobamide and a [4Fe-4S]2+/1+ center, both of which undergo redox cycling during catalysis, and that the benzimidazole base is uncoordinated to the cobalt (base off) in all three redox states, 3+, 2+, and 1+ [Ragsdale, S.W., Lindahl, P.A., & Münck, E. (1987) J. Biol. Chem. 262, 14289-14297]. In this paper, we have determined the midpoint reduction potentials for the metal centers in this C/Fe-SP by electron paramagnetic resonance and UV-visible spectroelectrochemical methods. The midpoint reduction potentials for the Co3+/2+ and the Co2+/1 couples of the corrinoid were found to be 300-350 and -504 mV (+/- 3 mV) in Tris-HCl at pH 7.6, respectively. We also removed the (5-methoxybenzimidazolyl)cobamide cofactor from the C/Fe-SP and determined that its Co3+/2+ reduction potential is 207 mV at pH 7.6. The midpoint potential for the [4Fe-4S]2+/1+ couple in the C/Fe-SP was determined to be -523 mV (+/- 5 mV). Removal of this cluster totally inactivates the protein; however, there is little effect of cluster removal on the midpoint potential of the Co2+/1+ couple. In addition, removal of the cobamide has an insignificant effect on the midpoint reduction potential of the [4Fe-4S] cluster. A 27-kDa corrinoid protein (CP) also was studied since it contains (5-methoxybenzimidazolyl)cobamide in the base-on form.(ABSTRACT TRUNCATED AT 250 WORDS)     

The β-ligands

The 5-hydroxybenzimidazolylcobamide (B₁₂-HBI) derivatives from Methanosarcina barkeri were isolated. SO₃^2?, CN^?, H₂O, NH₃ and CH₃^? were identified as β-ligands. Two B₁₂-HBI compounds with unidentified β-ligands were found, of which one constituted a major part of the corrinoid content. 5′-Deoxyadenosyl was found as a β-ligand of a corrinoid without α-ligand. Biosynthesis of CH₃B₁₂HBI was observed in cell-free extracts and depended on methanol and ATP.     

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.     

The Pseudoenzyme PDX1.2 Boosts Vitamin B6 Biosynthesis under Heat and Oxidative Stress in Arabidopsis

Vitamin B₆ is an indispensable compound for survival, well known as a cofactor for numerous central metabolic enzymes and more recently for playing a role in several stress responses, particularly in association with oxidative stress. Regulatory aspects for the use of the vitamin in these roles are not known. Here we show that certain plants carry a pseudoenzyme (PDX1.2), which is involved in regulating vitamin B₆ biosynthesis de novo under stress conditions. Specifically, we demonstrate that Arabidopsis PDX1.2 enhances the activity of its catalytic paralogs by forming a heterododecameric complex. PDX1.2 is strongly induced by heat as well as singlet oxygen stress, concomitant with an enhancement of vitamin B₆ production. Analysis of pdx1.2 knockdown lines demonstrates that boosting vitamin B₆ content is dependent on PDX1.2, revealing that this pseudoenzyme acts as a positive regulator of vitamin B₆ biosynthesis during such stress conditions in plants.     

Syntheses and characterization of vitamin B<Subscript>12</Subscript>–Pt(II) conjugates and their adenosylation in an enzymatic assay

Aiming at the use of vitamin B₁₂ as a drug delivery carrier for cytotoxic agents, we have reacted vitamin B₁₂ with trans-[PtCl(NH₃)₂(H₂O)]⁺, [PtCl₃(NH₃)]⁻ and [PtCl₄]²⁻. These Pt(II) precursors coordinated directly to the Co(III)-bound cyanide, giving the conjugates [{Co}–CN–{trans-PtCl(NH₃)₂}]⁺ (5), [{Co}–CN–{trans-PtCl₂(NH₃)}] (6), [{Co}–CN–{cis-PtCl₂(NH₃)}] (7) and [{Co}–CN–{PtCl₃}]⁻ (8) in good yields. Spectroscopic analyses for all compounds and X-ray structure elucidation for 5 and 7 confirmed their authenticity and the presence of the central “Co–CN–Pt” motif. Applicability of these heterodinuclear conjugates depends primarily on serum stability. Whereas 6 and 8 transmetallated rapidly to bovine serum albumin proteins, compounds 5 and 7 were reasonably stable. Around 20% of cyanocobalamin could be detected after 48 h, while the remaining 80% was still the respective vitamin B₁₂ conjugates. Release of the platinum complexes from vitamin B₁₂ is driven by intracellular reduction of Co(III) to Co(II) to Co(I) and subsequent adenosylation by the adenosyltransferase CobA. Despite bearing a rather large metal complex on the β-axial position, the cobamides in 5 and 7 are recognized by the corrinoid adenosyltransferase enzyme that catalyzes the formation of the organometallic C–Co bond present in adenosylcobalamin after release of the Pt(II) complexes. Thus, vitamin B₁₂ can potentially be used for delivering metal-containing compounds into cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Occurrence of corrinoid-containing membrane proteins in anaerobic bacteria

In Methanobacterium thermoautotrophicum a corrinoid-carrying membrane protein complex has been found, to which a tentative role in methane formation has been ascribed. To test this hypothesis representatives from different orders of methanogenic bacteria were examined for membrane-bound cobamides. These species differed in cell carbon precursor, in methane precursor, in occurrence of cytochromes and of the enzyme CO dehydrogenase, and in the systematic position (Methanobacteriales, Methanomicrobiales). All methanogenic bacteria contained cobamides in the membranes in amounts of about 60 nmol/g cell dry weight, in addition to different amounts of cobamides in the soluble cell fraction. The only central metabolic reaction obviously common to all of these methanogens was methyl coenzyme M reduction to CH₄. It is concluded that the membrane corrinoid participates in this energy-conserving reaction.Sulfate-reducing and acetogenic bacteria were included in this survey. They contained different amounts of cobamides in the soluble cell fraction but not in the membrane, a possible exception being Acetobacterium woodii.     

Redox potential changes during ATP‐dependent corrinoid reduction determined by redox titrations with europium(II)–DTPA

Corrinoids are essential cofactors of enzymes involved in the C₁ metabolism of anaerobes. The active, super‐reduced [Co^I] state of the corrinoid cofactor is highly sensitive to autoxidation. In O‐demethylases, the oxidation to inactive [Co^II] is reversed by an ATP‐dependent electron transfer catalyzed by the activating enzyme (AE). The redox potential changes of the corrinoid cofactor, which occur during this reaction, were studied by potentiometric titration coupled to UV/visible spectroscopy. By applying europium(II)–diethylenetriaminepentaacetic acid (DTPA) as a reductant, we were able to determine the midpoint potential of the [Co^II]/[Co^I] couple of the protein‐bound corrinoid cofactor in the absence and presence of AE and/or ATP. The data revealed that the transfer of electrons from a physiological donor to the corrinoid as the electron‐accepting site is achieved by increasing the potential of the corrinoid cofactor from ?530 ± 15 mV to ?250 ± 10 mV (E_SHE, pH 7.5). The first 50 to 100 mV of the shift of the redox potential seem to be caused by the interaction of nucleotide‐bound AE with the corrinoid protein or its cofactor. The remaining 150–200 mV had to be overcome by the chemical energy of ATP hydrolysis. The experiments revealed that Eu(II)–DTPA, which was already known as a powerful reducing agent, is a suitable electron donor for titration experiments of low‐potential redox centers. Furthermore, the results of this study will contribute to the understanding of thermodynamically unfavorable electron transfer processes driven by the power of ATP hydrolysis.     

Utilization of Hydrocarbons and Vitamin B12 Production by Bacillus badius

The accumulation of vitamin B₁₂ by Bacillus badins grown on hydrocarbon was investigated. The bacterium could assimilate n-alkanes of C₁₁–C₁₈, ethanol, fumarate, α-ketoglutarate and malate. n-Alkanes of C₁₆–C₁₈, were the best for vitamin B₁₂ production. The bacterium utilized well all of the nitrogen sources tested. Above all, ammonium dihydrogen phosphate was the best for the bacteria] growth and vitamin B₁₂ production. Addition of organic nutrients such as malt extract and meat extract, and addition of metal ions such as ferrous and cobalt promoted the growth and vitamin B₁₂ production. Interestingly, vitamin B₁₂ was produced mostly in the supernatant. The cyanoform of the corrinoid predominantly formed in the supernatant would confirm the identity with cobalamin.