Coenzyme B12-dependent enzymes in potatoes: Leucine 2,3-aminomutase and methylmalonyl-coa mutase

Extracts of potato tubers contain endogenous activities of two coenzyme B₁₂-dependent enzymes, leucine 2,3-aminomutase and methylmalonyl-CoA mutase. These activities are stimulated by the addition of coenzyme B₁₂ and are inhibited by intrinsic factor. The inhibition is overcome by coenzyme B₁₂.     

Magnetic field effects on coenzyme B12-dependent enzymes: Validation of ethanolamine ammonia lyase results and extension to human methylmalonyl CoA mutase

Enzymes with radical-pair intermediates have been considered as a likely target for purported magnetic field effects in humans. The bacterial enzyme ethanolamine ammonia lyase and the human enzyme methylmalonyl-CoA mutase catalyze coenzyme B₁₂-dependent rearrangement reactions. A common step in the mechanism of these two enzymes is postulated to be homolysis of the cobalt-carbon bond of the cofactor to generate a spin-correlated radical pair consisting of the 5′-deoxyadenosyl radical and cob(II)alamin [Ado^· Cbl(II)]. Thus, the reactions catalyzed by these enzymes are expected to be sensitive to an applied magnetic field according to the same principles that control radical pair chemical reactions. The magnetic field effect on ethanolamine ammonia lyase reported previously has been corroborated independently in one of the authors' laboratory. However, neither the human nor the bacterial mutase from Propionibacterium shermanii exhibits a magnetic field effect that could be greater than about 15%, considering the error limit imposed by the uncertainty of the coupled assay. Our studies suggest that putative magnetic field effects on physiological processes are not likely to be mediated by methylmalonyl-CoA mutase. Bioelectromagnetics 18:506–513, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Structural Basis of the Stereospecificity of Bacterial B12-dependent 2-Hydroxyisobutyryl-CoA Mutase

Bacterial coenzyme B₁₂-dependent 2-hydroxyisobutyryl-CoA mutase (HCM) is a radical enzyme catalyzing the stereospecific interconversion of (S)-3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA. It consists of two subunits, HcmA and HcmB. To characterize the determinants of substrate specificity, we have analyzed the crystal structure of HCM from Aquincola tertiaricarbonis in complex with coenzyme B₁₂ and the substrates (S)-3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA in alternative binding. When compared with the well studied structure of bacterial and mitochondrial B₁₂-dependent methylmalonyl-CoA mutase (MCM), HCM has a highly conserved domain architecture. However, inspection of the substrate binding site identified amino acid residues not present in MCM, namely HcmA Ile^A90 and Asp^A117. Asp^A117 determines the orientation of the hydroxyl group of the acyl-CoA esters by H-bond formation, thus determining stereospecificity of catalysis. Accordingly, HcmA D117A and D117V mutations resulted in significantly increased activity toward (R)-3-hydroxybutyryl-CoA. Besides interconversion of hydroxylated acyl-CoA esters, wild-type HCM as well as HcmA I90V and I90A mutant enzymes could also isomerize pivalyl- and isovaleryl-CoA, albeit at >10 times lower rates than the favorite substrate (S)-3-hydroxybutyryl-CoA. The nonconservative mutation HcmA D117V, however, resulted in an enzyme showing high activity toward pivalyl-CoA. Structural requirements for binding and isomerization of highly branched acyl-CoA substrates such as 2-hydroxyisobutyryl- and pivalyl-CoA, possessing tertiary and quaternary carbon atoms, respectively, are discussed.     

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.     

Metabolism of Glutamate in Purple Nonsulfur Bacteria Participation of Vitamin B12

Purple nonsulfur bacteria, Rhodospirillum rubrum and Rhodopseudomonas spheroides were found to possess coenzyme B₁₂-dependent glutamate mutase activity. Cell-free extracts of these bacteria grown on Co²⁺-containing media catalyzed the conversion of glutamate to β-methylaspartate and further to mesaconate. The activity of the cell-free extracts of these organisms cultivated on Co²⁺-deficient media was markedly lower than that of the normal cells. Addition of coenzyme B₁₂ to the former reaction mixture enhanced the mesaconate formation via β-methylaspartate. These results indicate the involvement of coenzyme Independent glutamate mutase of these bacteria in the dissimilation of glutamate to acetyl-CoA and pyruvate through the following pathway.

glutamate→β→methylaspartate→mesaconate→citramalate→→acetyl-CoA, pyruvate On the other hand, a greater part of glutamate was converted to α-hydroxyglutarate and succinate with the cell-free extracts of these photosynthetic bacteria. This fact, taking account of the presence of propionyl-CoA carboxylase in these bacteria, implies the participation of coenzyme B₁₂-dependent (R)-methylmalonyl-CoA mutase in the formation of succinate via the following route.

glutamate→α-ketoglutarate→α-hydroxyglutarate→propionate→propionyl-CoA→(S)-methylmalonyl-CoA→(R)-methylmalonyl-CoA→succinyl-CoA     

Vitamin B(12) incorporated with multiwalled carbon nanotube composite film for the determination of hydrazine

Electrochemically active composite film containing multiwalled carbon nanotubes (MWCNTs) and vitamin B₁₂ was synthesized on glassy carbon, gold, and indium tin oxide electrodes by the potentiodynamic method. The presence of MWCNTs in the composite film (MWCNT–B₁₂) modified electrode mediates vitamin B₁₂’s redox reaction, whereas vitamin B₁₂’s redox reaction does not occur at bare electrode. The electrochemical impedance spectroscopy studies reveal that MWCNTs present in MWCNT–B₁₂ film enhance electron shuttling between the reactant and electrode surface. The surface morphology of bare electrode, MWCNT film. and MWCNT–B₁₂ composite film was studied using atomic force microscopy, which reveals vitamin B₁₂ incorporated with MWCNTs. The MWCNT–B₁₂ composite film exhibits promising enhanced electrocatalysis toward hydrazine. The electrocatalysis response of hydrazine at MWCNT film and MWCNT–B₁₂ composite film was measured using cyclic voltammetry and amperometric current–time (i–t) curve techniques. The linear concentration range of hydrazine obtained at MWCNT–B₁₂ composite film using the i–t curve technique is 2.0 μM–1.95 mM. Similarly, the sensitivity of MWCNT–B₁₂ composite film for hydrazine determination using the i–t curve technique is 1.32 mA mM⁻¹ cm⁻², and the hydrazine’s limit of detection at MWCNT–B₁₂ composite film is 0.7 μM.     

A F420-dependent NADP reductase in the extremely thermophilic sulfate-reducing Archaeoglobus fulgidus

Archaeoglobus fulgidus, a sulfate-reducing Archaeon with a growth temperature optimum of 83°C, uses the 5-deazaflavin coenzyme F₄₂₀ rather than pyridine nucleotides in catabolic redox processes. The organism does, however, require reduced pyridine nuclcotides for biosynthetic purposes. We describe here that the Archaeon contains a coenzyme F₄₂₀-dependent NADP reductase which links anabolism to catabolism. The highly thermostable enzyme was purfied 3600-fold by affinity chromatography to apparent homogeneity in a 60% yield. The native enzyme with an apparent molecular mass of 55 kDa was composed of only one type of subunit of apparent molecular mass of 28 kDa. Spectroscopic analysis of the enzyme did not reveal the presence of any chromophoric prosthetic group. The purified enzyme catalyzed the reversible reduction of NADP (apparent K _M 40 M) with reduced F₄₂₀ (apparent K _M 20M) with a specific activity of 660 U/mg (apparent V _max) at pH 8.0 (pH optimum) and 80°C (temperature optimum). It was specific for both coenzyme F₄₂₀ and NADP. Sterochemical investigations showed that the F₄₂₀-dependent NADP reductase was Si face specific with respect to C5 of F₄₂₀ and Si face specific with respect to C4 of NADP.Abbreviations F₄₂₀ coenzyme F₄₂₀ - F₄₂₀H₂ 1,5-dihydrocoenzyme F₄₂₀ - H₄MPT tetrahydromethanopterin - CH=H₄MPT N⁵, N¹⁰-methylenetetrahydromethanopterin - MFR methanofuran - HPLC high performance liquid chromatography - methylene-H₄MPT dehydrogenase N⁵, N¹⁰-methylenetetrahydromethanopterin dehydrogenase - 1 U = 1 mol/min     

Activation of diol dehydrase by formamidinium or guanidinium ion, polyatomic monovalent cations having sp2 nitrogen

Coenzyme B₁₂-dependent diol dehydrase was activated by formamidinium or guanidinium ion. These polyatomic monovalent cations having sp² hybrid atomic orbitals and trigonal orientation were much more effective in activating the enzyme than methylammonium ion, but less active than NH₄+ or K⁺. Formamidinium and guanidinium ions were also effective both in forming and maintaining the binding of coenzyme B₁₂ to the apoenzyme. There is a close relationship between the effectiveness in activating the enzyme and those in forming and maintaining the holoenzyme, suggesting that these polyatomic monovalent cations play the same role in the diol dehydrase system as alkali metal monovalent cation such as K⁺.     

Cobalamin metabolism in cultured human chorionic villus cells

Cobalamin (Cbl, vitamin B₁₂) metabolism was analyzed in cultures of human chorionic villus (CV) cells obtained at 9–10 weeks of gestation. CV cells were shown to synthesize transcobalamin II (TCII) and to possess a high affinity receptor for that molecule. The cells bound and internalized radioactive cyanocobalamin (CN[⁵⁷Co]Cbl) complexed to TCII. This internalized CN[⁵⁷Co]Cbl was found to be converted to both methylCbl and adenosylCbl, the two intracellular coenzyme forms of Cbl, and bound to the two known intracellular Cbl requiring enzymes, methionine synthase (MS) and methylmalonyl-CoA mutase. Both enzyme systems were found to be functional in the intact cell by demonstrating the incorporation of the radioactive label from both [¹⁴C]CH₃-tetrahydrofolate and [¹⁴C]propionate into acid insoluble products. MS activity was also detected in lysed cell material. CV cells were shown not to be auxotrophic for methionine since they were able to utilize homocysteine in place of methionine for cell division. Since CV cells are capable of performing many of the complex events associated with Cbl metabolism, it may be possible to use these cells to diagnose genetic defects of Cbl metabolism. © 1993 Wiley-Liss, Inc.     

Influence of Zn<Superscript>2+</Superscript>, Co<Superscript>2+</Superscript> and dimethylbenzimidazole on vitamin B<Subscript>12</Subscript> biosynthesis by <Emphasis Type="Italic">Pseudomonas denitrificans</Emphasis>

Previous research has confirmed that cobalt ion and dimethylbenzimidazole (DMBI) are the precursors of vitamin B₁₂ biosynthesis, and porphobilinogen synthase (PBG synthase) is a zinc-requiring enzyme. In this paper, the effects of Zn²⁺, Co²⁺ and DMBI on vitamin B₁₂ production by Pseudomonas denitrificans in shake flasks were studied. Present experimental results demonstrated that the addition of the above mentioned three components to the fermentation medium could significantly stimulate the biosynthesis of vitamin B₁₂. The concentrations of zinc sulphate, cobaltous chloride and DMBI in the fermentation medium were further optimized with rotatable orthogonal central composite design and statistical analysis by Data Processing System (DPS) software. As a result, vitamin B₁₂ production was increased from 69.36 ± 0.66 to 78.23 ± 0.92 μg/ml.     

Production of 3‐hydroxypropionic acid from glycerol by recombinant Klebsiella pneumoniae ΔdhaTΔyqhD which can produce vitamin B12 naturally

3‐Hydroxypropionic acid (3‐HP) is an important platform chemical that can be used to synthesize a range of chemical compounds. A previous study demonstrated that recombinant Escherichia coli stains can produce 3‐HP from glycerol in the presence of vitamin B₁₂ (coenzyme B₁₂), when overexpressed with a coenzyme B₁₂‐dependent glycerol dehydratase (DhaB) and an aldehyde dehydrogenase. The present study examined the production of 3‐HP in recombinant Klebsiella pneumoniae strains, which naturally synthesizes vitamin B₁₂ and does not require supplementation of the expensive vitamin. The NAD⁺‐dependent gamma‐glutamyl‐gamma‐aminobutyraldehyde dehydrogenase (PuuC) of K. pneumoniae alone or with its DhaB was overexpressed homologously, and two major oxidoreductases, DhaT and YqhD, were disrupted. Without vitamin B₁₂ addition, the recombinant K. pneumoniae ΔdhaTΔyqhD overexpressing PuuC could produce ～3.8 g/L 3‐HP in 12 h of flask culture. However, this was possible only under the appropriate aeration conditions; 1,3‐propanediol (1,3‐PDO) (instead of 3‐HP) was mainly produced when aeration was insufficient, whereas a very small amount of both 3‐HP and 1,3‐PDO were produced when aeration was too high. The production of a small amount of 3‐HP under improper aeration conditions was attributed to either slow NAD⁺ regeneration (under low aeration) or reduced vitamin B₁₂ synthesis (under high aeration). In a glycerol fed‐batch bioreactor experiment under a constant DO of 5%, the strain, K. pneumoniae ΔdhaTΔyqhD, overexpressing both PuuC and DhaB could produce >28 g/L 3‐HP in 48 h with a yield of >40% on glycerol. Only small amount of 3‐HP was produced when cultivation was carried out at a constant aeration of 1 vvm or constant 10% DO. These results show that K. pneumoniae is potentially useful for the production of 3‐HP in an economical culture medium that does not require vitamin B₁₂. The results also suggest that the aeration conditions should be optimized carefully for the efficient production of 3‐HP while using this strain. Biotechnol. Bioeng. 2013; 110: 511–524. © 2012 Wiley Periodicals, Inc.     

Studies on the vitamin B12 auxotrophy of Rhodocyclus purpureus and two other vitamin B12-requiring purple nonsulfur bacteria

The vitamin B₁₂ requirement of Rhodocyclus purpureus 6770, Rhodospirillum tenue 1/67, and Rhodopseudomonas palustris G 53/2 was determined. A wide variety of biogenetic precursors of the vitamin including cobinamide, cobyric acid, cobinic acid and several partially amidated cobyrinic acids showed growth-promoting activity in all three strains. In R. purpureus vitamin B₁₂ could even be substituted by cobyrinic acid which is the first cobalt-containing precursor of vitamin B₁₂ so far established. Neither methionine, deoxynucleosides, dimethylbenzimidazole nor increased amounts of cobalt could replace vitamin B₁₂ as growth factor.Cupribalamin, which is a strong antimetabolite of vitamin B₁₂ in Escherichia coli 113-3 and Lactobacillus leichmannii ATCC 7830, exhibited only a weak antagonistic effect on growth of R. purpureus and R. tenue. Growth of R. palustris was not inhibited by cupribalamin. The cells of all three strains were shown to contain metal-free corrinoids in addition to cobalt-containing corrinoids. The principal products were identified as 5-deoxyadenosylcobalamin and hydrogenobalamin, the metal free analogue of vitamin B₁₂. The latter does not originate from the vitamin by removal of cobalt but is de novo biosynthesized as could be demonstrated in the case of R. purpureus by a labelling experiment with [¹³C] methyl-l-methionine.     

Guanylcobamide and hypoxanthylcobamide-Corrinoids formed by Desulfovibrio vulgaris

The corrinoids synthesized by the sulfate-reducing bacterium Desulfovibrio vulgaris were analyzed. The compounds found were guanylcobamide and hypoxanthylcobamide; structures were determined by mass spectrometry, ¹H-NMR, and ultraviolet/visible spectroscopy. D. vulgaris used externally added guanine to form guanylcobamide, as demonstrated with 8-¹⁴C-guanine. Addition of adenine did not lead to the formation of adenylcobamide (pseudovitamin B₁₂), whereas 5,6-dimethylbenzimidazole was transformed into vitamin B₁₂.     

Vitamin B12-dependent Methionine Synthetase in Photosynthetic Bacteria Partial Purification and Properties

Vitamin B₁₂-dependent methionine synthetase (N⁵-methyItetrahydrofolate-homocysteine Bi2-methyltransferase; EC 2.1.1.13) was partially purified from two different types of photo-synthetic bacteria, Chromatium D and Rhodospirillum rubrum.

Chromatium D, which does not produce vitamin B₁₂, possessed apomethionine synthetase when grown in the absence of the vitamin. Partially purified apoenzyme was converted to holoenzyme efficiently with CH₃B₁₂ or OHB₁₂. Holo-methionine synthetase was purified 244 fold with 56.4 % recovery from Chromatium D cells grown with vitamin B₁₂ added. The partially purified enzyme required reductants but was only partially dependent on S-adenosylmethionine.

On the other hand, Rsp. rubrum methionine synthetase which was always present as holoenzyme, in contrast with that of Chromatium D, was purified 40 fold with 2.8% recovery. The obtained preparation required S-adenosylmethionine and reductants for the enzyme activity. The optimal pH of Chromatium D enzyme and of Rsp. rubrum enzyme was in the range of 7.5~7.8 and 6.5~6.75, respectively.     

Methyl-coenzyme M reductase and other enzymes involved in methanogenesis from CO2 and H2 in the extreme thermophile Methanopyrus kandleri

Methanopyrus kandleri belongs to a novel group of abyssal methanogenic archaebacteria that can grow at 110°C on H₂ and CO₂ and that shows no close phylogenetic relationship to any methanogen known so far. Methyl-coenzyme M reductase, the enzyme catalyzing the methane forming step in the energy metabolism of methanogens, was purified from this hyperthermophile. The yellow protein with an absorption maximum at 425 nm was found to be similar to the methyl-coenzyme M reductase from other methanogenic bacteria in that it was composed each of two -, - and -subunits and that it contained the nickel porphinoid coenzyme F₄₃₀ as prosthetic group. The purified reductase was inactive. The N-terminal amino acid sequence of the -subunit was determined. A comparison with the N-terminal sequences of the -subunit of methyl-coenzyme M reductases from other methanogenic bacteria revealed a high degree of similarity.Besides methyl-coenzyme M reductase cell extracts of M. kandleri were shown to contain the following enzyme activities involved in methanogenesis from CO₂ (apparent V_max at 65°C): formylmethanofuran dehydrogenase, 0.3 U/mg protein; formyl-methanofuran: tetrahydromethanopterin formyltransferase, 13 U/mg; N ⁵,N¹⁰-methenyltetrahydromethanopterin cyclohydrolase, 14 U/mg; N ⁵,N¹⁰-methylenetetrahydromethanopterin dehydrogenase (H₂-forming), 33 U/mg; N ⁵,N¹⁰-methylenetetrahydromethanopterin reductase (coenzyme F₄₂₀ dependent), 4 U/mg; heterodisulfide reductase, 2 U/mg; coenzyme F₄₂₀-reducing hydrogenase, 0.01 U/mg; and methylviologen-reducing hydrogenase, 2.5 U/mg. Apparent K_m values for these enzymes and the effect of salts on their activities were determined.The coenzyme F₄₂₀ present in M. kandleri was identified as coenzyme F₄₂₀-2 with 2 -glutamyl residues.Abbreviations H–S-CoM coenzyme M - CH₃–S-CoM methylcoenzyme M - H–S-HTP 7-mercaptoheptanoylthreonine phosphate - MFR methanofuran - CHO-MFR formyl-MFR - H₄MPT tetrahydromethanopterin - CHO–H₄MPT N ⁵-formyl-H₄MPT - CH=H₄MPT⁺ N ⁵,N¹⁰-methenyl-H₄MPT - CH₂=H₄MPT N ⁵,N¹⁰-methylene-H₄MPT - CH₃–H₄MPT N ⁵-methyl-H₄MPT - F₄₂₀ coenzyme F₄₂₀ - 1 U= 1 mol/min     

Conditions for the production of Agrocin 84 byAgrobacterium radiobacter K84

Summary A chemically defined medium was developed that supported the growth ofAgrobacterium radiobacter K84 and the production of agrocin 84. Various supplements were investigated for their effect on growth rate and production of agrocin 84 using a well-diffusion assay method. Mannitol was found to be a better substrate for growth ofA. radiobacter K84 compared to the other sugar alcohols and sugars tested. By contrast,d-fructose was the better substrate for the production of agrocin 84. Biotin supplementation stimulated production of agrocin 84 but did not eliminate the diauxic lag seen with the basal medium. The opine, octopine, inhibited growth ofA. radiobacter K84 and production of agrocin 84 as did coenzyme B₁₂. By contrast, the cytokinin, isopentenyl adenosine, was marginally stimulatory to production, as was vitamin B₁₂. Acetosyringone supplementation had a negligible effect on growth rate and production of agrocin 84.     

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.     

EFFECT OF HORMONES AND VITAMIN B12 ON GAMETOPHORE DEVELOPMENT IN THE MOSS PYLAISIELLA SELWYNII

Bud formation in the moss Pylaisiella selwynii is greatly enhanced by cytokinins at concentrations as low as 10⁻¹²m , yet these buds usually fail to develop into normal gametophores. Various ratios at different concentrations of the cytokinin N-6-γ,γ-dimethylallylaminopurine to indoleacetic acid failed to enhance bud initiation over that obtained with cytokinin alone or to permit normal gametophore development. Deletion of the cobaltous ions from the culture medium prevented the appearance of the few gametophores usually formed in the complete medium, but different amounts of cobaltous ion did not significantly enhance initiation of gametophore development. Bud initiation was enhanced 3- to 20-fold by vitamin B₁₂ at 10⁻⁵m or by B₁₂ coenzyme at 10⁻⁴m , and the time of appearance of these buds was advanced by 6–12 days compared to control plants. At these concentrations of the B₁₂ compounds the buds formed normal gametophores, but at 10⁻⁴m vitamin B₁₂ they grew into callus-like masses similar to those obtained with cytokinins. Although the effects of B₁₂ on bud initiation and development mimicked those of cytokinins, except in permitting normal development, no additive or synergistic effects were observed when they were tested together. It is suggested that B₁₂ may play a regulatory role in the control of gametophore initiation and development in mosses.     

Metabolic engineering of cobalamin (vitamin B12) production in Bacillus megaterium

Cobalamin (vitamin B₁₂) production in Bacillus megaterium has served as a model system for the systematic evaluation of single and multiple directed molecular and genetic optimization strategies. Plasmid and genome-based overexpression of genes involved in vitamin B₁₂ biosynthesis, including cbiX, sirA, modified hemA, the operons hemAXCDBL and cbiXJCDETLFGAcysG^AcbiYbtuR, and the regulatory gene fnr, significantly increased cobalamin production. To reduce flux along the heme branch of the tetrapyrrole pathway, an antisense RNA strategy involving silencing of the hemZ gene encoding coproporphyrinogen III oxidase was successfully employed. Feedback inhibition of the initial enzyme of the tetrapyrrole biosynthesis, HemA, by heme was overcome by stabilized enzyme overproduction. Similarly, the removal of the B₁₂ riboswitch upstream of the cbiXJCDETLFGAcysG^AcbiYbtuR operon and the recombinant production of three different vitamin B₁₂ binding proteins (glutamate mutase GlmS, ribonucleotide triphosphate reductase RtpR and methionine synthase MetH) partly abolished B₁₂-dependent feedback inhibition. All these strategies increased cobalamin production in B. megaterium. Finally, combinations of these strategies enhanced the overall intracellular vitamin B₁₂ concentrations but also reduced the volumetric cellular amounts by placing the organism under metabolic stress.