Human ATP:Cob(I)alamin adenosyltransferase and its interaction with methionine synthase reductase

The final step in the conversion of vitamin B(12) into coenzyme B(12) (adenosylcobalamin, AdoCbl) is catalyzed by ATP:cob(I)alamin adenosyltransferase (ATR). Prior studies identified the human ATR and showed that defects in its encoding gene underlie cblB methylmalonic aciduria. Here two common polymorphic variants of the ATR that are found in normal individuals are expressed in Escherichia coli, purified, and partially characterized. The specific activities of ATR variants 239K and 239M were 220 and 190 nmol min(-1) mg(-1), and their K(m) values were 6.3 and 6.9 mum for ATP and 1.2 and 1.6 mum for cob(I)alamin, respectively. These values are similar to those obtained for previously studied bacterial ATRs indicating that both human variants have sufficient activity to mediate AdoCbl synthesis in vivo. Investigations also showed that purified recombinant human methionine synthase reductase (MSR) in combination with purified ATR can convert cob(II)alamin to AdoCbl in vitro. In this system, MSR reduced cob(II)alamin to cob(I)alamin that was adenosylated to AdoCbl by ATR. The optimal stoichiometry for this reaction was approximately 4 MSR/ATR and results indicated that MSR and ATR physically interacted in such a way that the highly reactive reaction intermediate [cob(I)alamin] was sequestered. The finding that MSR reduced cob(II)alamin to cob(I)alamin for AdoCbl synthesis (in conjunction with the prior finding that MSR reduced cob(II)alamin for the activation of methionine synthase) indicates a dual physiological role for MSR.     

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.     

In vivo expression of human ATP:cob(I)alamin adenosyltransferase (ATR) using recombinant adeno-associated virus (rAAV) serotypes 2 and 8

BACKGROUND: Methylmalonic aciduria (MMA) is an autosomal recessive disease with symptoms that include ketoacidosis, lethargy, recurrent vomiting, dehydration, respiratory distress, muscular hypotonia and death due to methylmalonic acid levels that are up to 1000-fold greater than normal. CblB MMA, a subset of the mutations leading to MMA, is caused by a deficiency in the enzyme cob(I)alamin adenosyltransferase (ATR). No animal model currently exists for this disease. ATR functions within the mitochondria matrix in the final conversion of cobalamin into coenzyme B(12), adenosylcobalamin (AdoCbl). AdoCbl is a required coenzyme for the mitochondrial enzyme methylmalonyl-CoA mutase (MCM). METHODS: The human ATR cDNA was cloned into a recombinant adeno-associated virus (rAAV) vector and packaged into AAV 2 or 8 capsids and delivered by portal vein injection to C57/Bl6 mice at a dose of 1 x 10(10) and 1 x 10(11) particles. Eight weeks post-injection RNA, genomic DNA and protein were then extracted and analyzed. RESULTS: Using primer pairs specific to the cytomegalovirus (CMV) enhancer/chicken beta-actin (CBAT) promoter within the rAAV vectors, genome copy numbers were found to be 0.03, 2.03 and 0.10 per cell in liver for the rAAV8 low dose, rAAV8 high dose and rAAV2 high dose, respectively. Western blotting performed on mitochondrial protein extracts demonstrated protein levels were comparable to control levels in the rAAV8 low dose and rAAV2 high dose animals and 3- to 5-fold higher than control levels were observed in high dose animals. Immunostaining demonstrated enhanced transduction efficiency of hepatocytes to over 40% in the rAAV8 high dose animals, compared to 9% and 5% transduction in rAAV2 high dose and rAAV8 low dose animals, respectively. CONCLUSIONS: These data demonstrate the feasibility of efficient ATR gene transfer to the liver as a prelude to future gene therapy experiments.     

Identification of the human and bovine ATP:Cob(I)alamin adenosyltransferase cDNAs based on complementation of a bacterial mutant

In humans, deficiencies in coenzyme B12-dependent methylmalonyl-CoA mutase (MCM) lead to methylmalonyl aciduria, a rare disease that is often fatal in newborns. Such deficiencies can result from inborn errors in the MCM structural gene or from mutations that impair the assimilation of dietary cobalamins into coenzyme B12 (Ado-B12), the required cofactor for MCM. ATP:cob(I)alamin adenosyltransferase (ATR) catalyzes the terminal step in the conversion of cobalamins into Ado-B12. Substantial evidence indicates that inherited defects in this enzyme lead to methylmalonyl aciduria, but the corresponding ATR gene has not been identified. Here we report the identification of the bovine and human ATR cDNAs as well as the corresponding human gene. A bovine liver cDNA expression library was screened for clones that complemented an ATR-deficient bacterial strain for color formation on aldehyde indicator medium, and four positive clones were isolated. The DNA sequences of two clones were determined and found to be identical. Sequence similarity searching was then used to identify a homologous human cDNA (89% identity) and its corresponding gene that is located on chromosome XII. The bovine and human cDNAs were independently cloned and expressed in Escherichia coli. Enzyme assays showed that expression strains produced 87 and 98 nmol/min/mg ATR activity, respectively. These specific activities are in line with values reported previously for bacterial ATR enzymes. Subsequent studies showed that the human cDNA clone complemented an ATR-deficient bacterial mutant for Ado-B12-dependent growth on 1,2-propanediol. This demonstrated that the human ATR is active under physiological conditions albeit in a heterologous host. In addition, Western blots were used to show that ATR expression is altered in cell lines derived from cblB methylmalonyl aciduria patients compared with cell lines from normal individuals. We propose that inborn errors in the human ATR gene identified here result in methylmalonyl aciduria. The identification of genes involved in this disorder will allow improvements in the diagnosis and treatment of this serious disease.     

Multiple roles of ATP:cob(I)alamin adenosyltransferases in the conversion of B12 to coenzyme B12

Our mechanistic understanding of the conversion of vitamin B₁₂ into coenzyme B₁₂ (a.k.a. adenosylcobalamin, AdoCbl) has been substantially advanced in recent years. Insights into the multiple roles played by ATP:cob(I)alamin adenosyltransferase (ACA) enzymes have emerged through the crystallographic, spectroscopic, biochemical, and mutational analyses of wild-type and variant proteins. ACA enzymes circumvent the thermodynamic barrier posed by the very low redox potential associated with the reduction of cob(II)alamin to cob(I)alamin by generating a unique four-coordinate cob(II)alamin intermediate that is readily converted to cob(I)alamin by physiological reductants. ACA enzymes not only synthesize AdoCbl but also they deliver it to the enzymes that use it, and in some cases, enzymes in which its function is needed to maintain the fidelity of the AdoCbl delivery process have been identified. Advances in our understanding of ACA enzyme function have provided valuable insights into the role of specific residues, and into why substitutions of these residues have profound negative effects on human health. From an applied science standpoint, a better understanding of the adenosylation reaction may lead to more efficient ways of synthesizing AdoCbl.     

New perspectives for pharmacological chaperoning treatment in methylmalonic aciduria cblB type

Methylmalonic aciduria cblB type (MMA cblB) is caused by the impairment of ATP:cob(I)alamin adenosyltransferase (ATR), the enzyme responsible for the synthesis of adenosylcobalamin (AdoCbl) from cob(I)alamin. No definitive treatment is available for patients with this condition and novel therapeutic strategies are therefore much needed. Recently, we described a proof-of-concept regarding the use of pharmacological chaperones as a treatment. This work describes the effect of two potential pharmacological chaperones - compound V (N-{[(4-chlorophenyl)carbamothioyl]amino}-2-phenylacetamide) and compound VI (4-(4-(4-fluorophenyl)-5-methyl-1H-pyrazol-3-yl)benzene-1,3-diol) - on six ATR mutants, including the most common, p.Arg186Trp. Comprehensive functional analysis identified destabilizing (p.Arg186Gln, p.Arg190Cys, p.Arg190His, p.Arg191Gln and p.Glu193Lys) and oligomerization (p.Arg186Trp and p.Arg191Gln) mutations. In a cellular model overexpressing the destabilizing/oligomerization mutations, compounds V and VI had a positive effect on the stability and activity of all ATR variants. When provided in combination with hydroxocobalamin a more positive effect was obtained than with the compounds alone, even in mutations previously described as B₁₂ non-responsive. In addition, a normal oligomerization profile was recovered after treatment of the p.Arg186Trp mutant with both compounds. These promising results confirm MMA cblB type as a conformational disorder and hence, pharmacological chaperones as a new therapeutic option alone or in combination with hydroxocobalamin for many patients with MMA cblB.     

Binding of Cob(II)alamin to the adenosylcobalamin-dependent ribonucleotide reductase from Lactobacillus leichmannii. Identification of dimethylbenzimidazole as the axial ligand

The ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii catalyzes the reduction of nucleoside 5'-triphosphates to 2'-deoxynucleoside 5'-triphosphates and uses coenzyme B12, adenosylcobalamin (AdoCbl), as a cofactor. Use of a mechanism-based inhibitor, 2'-deoxy-2'-methylenecytidine 5'-triphosphate, and isotopically labeled RTPR and AdoCbl in conjunction with EPR spectroscopy has allowed identification of the lower axial ligand of cob(II)alamin when bound to RTPR. In common with the AdoCbl-dependent enzymes catalyzing irreversible heteroatom migrations and in contrast to the enzymes catalyzing reversible carbon skeleton rearrangements, the dimethylbenzimidazole moiety of the cofactor is not displaced by a protein histidine upon binding to RTPR.     

Hepatocyte-like cells differentiated from methylmalonic aciduria cblB type induced pluripotent stem cells: A platform for the evaluation of pharmacochaperoning

Methylmalonic aciduria cblB type (MMA cblB type, MMAB OMIM #251110), caused by a deficiency in the enzyme ATP:cob(I)alamin adenosyltransferase (ATR, E.C_2. 5.1.17), is a severe metabolic disorder with a poor prognosis despite treatment. We recently described the potential therapeutic use of pharmacological chaperones (PCs) after increasing the residual activity of ATR in patient-derived fibroblasts. The present work reports the successful generation of hepatocyte-like cells (HLCs) differentiated from two healthy and two MMAB induced pluripotent stem cell (iPSC) lines, and the use of this platform for testing the effects of PCs. The MMAB cells produced little ATR, showed reduced residual ATR activity, and had higher concentrations of methylmalonic acid compared to healthy HLCs. Differential proteome analysis revealed the two MMAB HCLs to show reproducible differentiation, but this was not so for the healthy HLCs. Interestingly, PC treatment in combination with vitamin B₁₂ increased the amount of ATR available, and subsequently ATR activity, in both MMAB HLCs. More importantly, the treatment significantly reduced the methylmalonic acid content of both. In summary, the HLC model would appear to be an excellent candidate for the pharmacological testing of the described PCs, for analyzing the effects of new drugs, and investigating the repurposing of older drugs, before testing in animal models.     

9-(Adenylyl)alkylcobalamins as inhibitors of adenosylcobalamin-dependent glycerol dehydratase from Aerobacter aerogenes

The behavior of two coenzyme analogs, [(5-aden-9-yl)methoxyethyl] cob (III) alamin and [(5-aden-9-yl)pentyl] cob (III) alamin modified at the nucleoside ligand sugar moiety was studied in the system of adenosyl-cobalamin-dependent glycerol dehydratase from Aerobacter aerogenes. It was shown that neither of the analogs possesses coenzyme properties and that both are strong competitive inhibitors for adenosylcobalamin (AdoCbl). The affinity of the two analogs for the apoenzyme is higher than that of AdoCbl. The data obtained are indicative of the essential role of the ribofuranoside fragment of AdoCbl in the manifestation of the coenzyme activity. The apoenzyme interaction with the analogs under study is discussed in terms of the Dreiding stereomodels for AdoCbl and its analogs.     

Regulation by SREBP-2 defines a potential link between isoprenoid and adenosylcobalamin metabolism

Mevalonate kinase (MVK) catalyses an early step in cholesterol biosynthesis converting mevalonate to phosphomevalonate. Cob(I)alamin adenosyltransferase (MMAB) converts cob(I)alamin to adenosylcobalamin, functionally required for mitochondrial methylmalonyl-CoA mutase activity and succinyl-CoA formation. These two synthenic genes are found in a head-to-head formation on chromosome 12 in man and chromosome 5 in mouse. The 330bp intergenic region showed several conserved NF-Y sites indicative of potential bidirectional regulatory SREBP synergism. Both MVK and MMAB appear to be regulated in a similar manner, to a large extent by SREBP-2, since their tissue expression pattern was similar and both genes were suppressed by an excess of cholesterol as well as SREBP-2 knockdown. Statin treatment in mice upregulated both Mvk and Mmab mRNA levels indicating that this treatment may be useful in inborn errors of cblB complementation associated with methylmalonic aciduria as well as hyper IgD and periodic fever syndrome (HIDS).     

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.     

Interaction of 3'-[3H]2'-Chloro-2'-deoxyuridine 5'-triphosphate with ribonucleotide reductase from Lactobacillus leichmannii

Incubation of [3'-3H]2'-chloro-2'-deoxyuridine 5'-triphosphate (CldUTP) with adenosylcobalamin (AdoCbl), reductant, and ribonucleotide reductase from Lactobacillus leichmannii results in the production of 3H2O, uracil, tripolyphosphate, 5'-deoxyadenosine, and cob(II)alamin. The rate of 3H2O release (0.19 mumol/min/mg) is almost identical with the rate of UTP reduction (0.24 mumol/min/mg). The amount of 3H2O release is dependent upon the enzyme to cofactor ratio. With a ribonucleotide reductase: AdoCbl ratio of 1:1000, approximately 500 eq of 3H2O are released. At this time the enzyme is still active, but further destruction of cofactor and turnover of CldUTP is prevented by competitive inhibition of Co(II) + 5'-deoxyadenosine with AdoCbl for binding to ribonucleotide reductase. The 5'-deoxyadenosine and AdoCbl reisolated during incubation of [3'-3H]CldUTP and ribonucleotide reductase contains no detectable radioactivity.     

The defect in the cblB class of human methylmalonic acidemia: Deficiency of cob(I)alamin adenosyltransferase activity in extracts of cultured fibroblasts

We show that the reductants present in the $\text{in}\text{vitro}$ assay used to measure the formation of adenosylcobalamin from cob(III)alamin by cell-free extracts of human fibroblasts result in the non-enzymatic reduction of cob(III)alamin to cob(I)alamin. Hence, the $\text{in}\text{vitro}$ assay uniquely estimates the activity of ATP:cob(I)alamin adenosyltransferase (EC 2.5.1.17). Based on additional studies with extracts of fibroblasts from patients in the $\text{cbl}\text{B}$ class of human methylmalonic acidemia and from their parents, we conclude that this mutant class results from a specific deficiency of adenosyltransferase activity which is inherited as an autosomal recessive trait.     

Cobalamin-dependent methionine synthase: probing the role of the axial base in catalysis of methyl transfer between methyltetrahydrofolate and exogenous cob(I)alamin or cob(I)inamide

Cobalamin-dependent methionine synthase (MetH) catalyzes the transfer of methyl groups between methyltetrahydrofolate (CH(3)-H(4)folate) and homocysteine, with the enzyme-bound cobalamin serving as an intermediary in the methyl transfers. An MetH fragment comprising residues 2-649 contains modules that bind and activate CH(3)-H(4)folate and homocysteine and catalyze methyl transfers to and from exogenous cobalamin. Comparison of the rates of reaction of cobalamin, which contains a dimethylbenzimidazole nucleotide coordinated to the cobalt in the lower axial position, and cobinamide, which lacks the dimethylbenzimidazole nucleotide, allows assessment of the degree of stabilization the dimethylbenzimidazole base provides for methyl transfer between CH(3)-H(4)folate bound to MetH(2-649) and exogenous cob(I)alamin. When the reactions of cob(I)alamin or cob(I)inamide with CH(3)-H(4)folate are compared, the observed second-order rate constants are 2.7-fold faster for cob(I)alamin; in the reverse direction, methylcobinamide reacts 35-fold faster than methylcobalamin with enzyme-bound tetrahydrofolate. These measurements can be used to estimate the influence of the dimethylbenzimidazole ligand on both the thermodynamics and kinetics of methyl transfer between methyltetrahydrofolate and cob(I)alamin or cob(I)inamide. The free energy change for methyl transfer from CH(3)-H(4)folate to cob(I)alamin is 2.8 kcal more favorable than that for methyl transfer to cob(I)inamide. Dimethylbenzimidazole contributes approximately 0.6 kcal/mol of stabilization for the forward reaction and approximately 2.2 kcal/mol of destabilization for the reverse reaction. Binding of methylcobalamin to full-length methionine synthase is accompanied by ligand substitution, and switching between "base-on" and "base-off" states of the cofactor has been demonstrated [Bandarian, V., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 8156-8163]. The present results disfavor a major role for such switching in catalysis of methyl transfer, and are consistent with the hypothesis that the primary role of the ligand triad in methionine synthase is controlling the distribution of enzyme conformations during catalysis.     

Reduction of Cob(III)alamin to Cob(II)alamin in Salmonella enterica serovar typhimurium LT2

Reduction of the cobalt ion of cobalamin from the Co(III) to the Co(I) oxidation state is essential for the synthesis of adenosylcobalamin, the coenzymic form of this cofactor. A cob(II)alamin reductase activity in Salmonella enterica serovar Typhimurium LT2 was isolated to homogeneity. N-terminal analysis of the homogeneous protein identified NAD(P)H:flavin oxidoreductase (Fre) (EC 1.6.8.1) as the enzyme responsible for this activity. The fre gene was cloned, and the overexpressed protein, with a histidine tag at its N terminus, was purified to homogeneity by nickel affinity chromatography. His-tagged Fre reduced flavins (flavin mononucleotide [FMN] and flavin adenine dinucleotide [FAD]) and cob(III)alamin to cob(II)alamin very efficiently. Photochemically reduced FMN substituted for Fre in the reduction of cob(III)alamin to cob(II)alamin, indicating that the observed cobalamin reduction activity was not Fre dependent but FMNH(2) dependent. Enzyme-independent reduction of cob(III)alamin to cob(II)alamin by FMNH(2) occurred at a rate too fast to be measured. The thermodynamically unfavorable reduction of cob(II)alamin to cob(I)alamin was detectable by alkylation of the cob(I)alamin nucleophile with iodoacetate. Detection of the product, caboxymethylcob(III)alamin, depended on the presence of FMNH(2) in the reaction mixture. FMNH(2) failed to substitute for potassium borohydride in in vitro assays for corrinoid adenosylation catalyzed by the ATP:co(I)rrinoid adenosyltransferase (CobA) enzyme, even under conditions where Fre and NADH were present in the reaction mixture to ensure that FMN was always reduced. These results were interpreted to mean that Fre was not responsible for the generation of cob(I)alamin in vivo. Consistent with this idea, a fre mutant displayed wild-type cobalamin biosynthetic phenotypes. It is proposed that S. enterica serovar Typhimurium LT2 may not have a cob(III)alamin reductase enzyme and that, in vivo, nonadenosylated cobalamin and other corrinoids are maintained as co(II)rrinoids by reduced flavin nucleotides generated by Fre and other flavin oxidoreductases.     

Probing the role of the histidine 759 ligand in cobalamin-dependent methionine synthase

Cobalamin-dependent methionine synthase (MetH) of Escherichia coli is a 136 kDa, modular enzyme that undergoes large conformational changes as it uses a cobalamin cofactor as a donor or acceptor in three separate methyl transfer reactions. At different points during the reaction cycle, the coordination to the cobalt of the cobalamin changes; most notably, the imidazole side chain of His759 that coordinates to the cobalamin in the "His-on" state can dissociate to produce a "His-off" state. Here, two distinct species of the cob(II)alamin-bound His759Gly variant have been identified and separated. Limited proteolysis with trypsin was employed to demonstrate that the two species differ in protein conformation. Magnetic circular dichroism and electron paramagnetic resonance spectroscopies were used to show that the two species also differ with respect to the axial coordination to the central cobalt ion of the cobalamin cofactor. One form appears to be in a conformation poised for reductive methylation with adenosylmethionine; this form was readily reduced to cob(I)alamin and subsequently methylated [albeit yielding a unique, five-coordinate methylcob(III)alamin species]. Our spectroscopic data revealed that this form contains a five-coordinate cob(II)alamin species, with a water molecule as an axial ligand to the cobalt. The other form appears to be in a catalytic conformation and could not be reduced to cob(I)alamin under any of the conditions tested, which precluded conversion to the methylcob(III)alamin state. This form was found to possess an effectively four-coordinate cob(II)alamin species that has neither water nor histidine coordinated to the cobalt center. The formation of this four-coordinate cob(II)alamin "dead-end" species in the His759Gly variant illustrates the importance of the His759 residue in governing the equilibria between the different conformations of MetH.     

Functional characterization and mutation analysis of human ATP:Cob(I)alamin adenosyltransferase

ATP:cob(I)alamin adenosyltransferase catalyzes the final step in the conversion of vitamin B 12 into the active coenzyme, adenosylcobalamin. Inherited defects in the gene for the human adenosyltransferase (hATR) result in methylmalonyl aciduria (MMA), a rare but life-threatening illness. In this study, we conducted a random mutagenesis of the hATR coding sequence. An ATR-deficient strain of Salmonella was used as a surrogate host to screen for mutations that impaired hATR activity in vivo. Fifty-seven missense mutations were isolated. These mapped to 30 positions of the hATR, 25 of which had not previously been shown to impair enzyme activity. Kinetic analysis and in vivo tests for enzyme activity were performed on the hATR variants, and mutations were mapped onto a hATR structural model. These studies functionally defined the hATR active site and tentatively implicated three amino acid residues in facilitating the reduction of cob(II)alamin to cob(I)alamin which is a prerequisite to adenosylation.     

Functional genomic, biochemical, and genetic characterization of the Salmonella pduO gene, an ATP:cob(I)alamin adenosyltransferase gene

Salmonella enterica degrades 1,2-propanediol by a pathway dependent on coenzyme B12 (adenosylcobalamin [AdoCb1]). Previous studies showed that 1,2-propanediol utilization (pdu) genes include those for the conversion of inactive cobalamins, such as vitamin B12, to AdoCbl. However, the specific genes involved were not identified. Here we show that the pduO gene encodes a protein with ATP:cob(I)alamin adenosyltransferase activity. The main role of this protein is apparently the conversion of inactive cobalamins to AdoCbl for 1,2-propanediol degradation. Genetic tests showed that the function of the pduO gene was partially replaced by the cobA gene (a known ATP:corrinoid adenosyltransferase) but that optimal growth of S. enterica on 1,2-propanediol required a functional pduO gene. Growth studies showed that cobA pduO double mutants were unable to grow on 1,2-propanediol minimal medium supplemented with vitamin B(12) but were capable of growth on similar medium supplemented with AdoCbl. The pduO gene was cloned into a T7 expression vector. The PduO protein was overexpressed, partially purified, and, using an improved assay procedure, shown to have cob(I)alamin adenosyltransferase activity. Analysis of the genomic context of genes encoding PduO and related proteins indicated that particular adenosyltransferases tend to be specialized for particular AdoCbl-dependent enzymes or for the de novo synthesis of AdoCbl. Such analyses also indicated that PduO is a bifunctional enzyme. The possibility that genes of unknown function proximal to adenosyltransferase homologues represent previously unidentified AdoCbl-dependent enzymes is discussed.     

The C-terminal domain of CblD interacts with CblC and influences intracellular cobalamin partitioning

Mutations in cobalamin or B₁₂ trafficking genes needed for cofactor assimilation and targeting lead to inborn errors of cobalamin metabolism. The gene corresponding to one of these loci, cblD, affects both the mitochondrial and cytoplasmic pathways for B₁₂ processing. We have demonstrated that fibroblast cell lines from patients with mutations in CblD, can dealkylate exogenously supplied methylcobalamin (MeCbl), an activity catalyzed by the CblC protein, but show imbalanced intracellular partitioning of the cofactor into the MeCbl and 5′-deoxyadenosylcobalamin (AdoCbl) pools. These results confirm that CblD functions downstream of CblC in the cofactor assimilation pathway and that it plays an important role in controlling the traffic of the cofactor between the competing cytoplasmic and mitochondrial routes for MeCbl and AdoCbl synthesis, respectively. In this study, we report the interaction of CblC with four CblD protein variants with variable N-terminal start sites. We demonstrate that a complex between CblC and CblD can be isolated particularly under conditions that permit dealkylation of alkylcobalamin by CblC or in the presence of the corresponding dealkylated and oxidized product, hydroxocobalamin (HOCbl). A weak CblC·CblD complex is also seen in the presence of cyanocobalamin. Formation of the CblC·CblD complex is observed with all four CblD variants tested suggesting that the N-terminal 115 residues missing in the shortest variant are not essential for this interaction. Furthermore, limited proteolysis of the CblD variants indicates the presence of a stable C-terminal domain spanning residues ∼116–296. Our results are consistent with an adapter function for CblD, which in complex with CblC·HOCbl, or possibly the less oxidized CblC·cob(II)alamin, partitions the cofactor between AdoCbl and MeCbl assimilation pathways.