Cob Alamin Adenosyltransferase Research Articles

Coordination Chemistry Controls Coenzyme B12 Synthesis by Human Adenosine Triphosphate:Cob(I)alamin Adenosyltransferase.

Cobalamin (or vitamin B12)-dependent enzymes and trafficking chaperones exploit redox-linked coordination chemistry to control the cofactor reactivity during catalysis and translocation. As the cobalt oxidation state decreases from 3+ to 1+, the preferred cobalamin geometry changes from six- to four-coordinate (4-c). In this study, we reveal the sizable thermodynamic gain that accrues for human adenosine triphosphate (ATP):cob(I)alamin adenosyltransferase (or MMAB) by enforcing an unfavorable 4-c cob(II)alamin geometry. MMAB-bound cob(II)alamin is reduced to the supernucleophilic cob(I)alamin intermediate during the synthesis of 5'-deoxyadenosylcobalamin. Herein, we report the first experimentally determined reduction potential for 4-c cob(II)alamin (-325 ± 9 mV), which is 180 mV more positive than for the five-coordinate (5-c) water-liganded species. The redox potential of MMAB-bound cob(II)alamin is within the range of adrenodoxin, which we demonstrate functions as an electron donor. We also show that stabilization of 5-c cob(II)alamin by a subset of MMAB patient variants compromises the reduction by adrenodoxin, explaining the underlying pathogenic mechanism.

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.

Zng1 is a GTP-dependent zinc transferase needed for activation of methionine aminopeptidase.

The evolution of zinc (Zn) as a protein cofactor altered the functional landscape of biology, but dependency on Zn also created an Achilles' heel, necessitating adaptive mechanisms to ensure Zn availability to proteins. A debated strategy is whether metallochaperones exist to prioritize essential Zn-dependent proteins. Here, we present evidence for a conserved family of putative metal transferases in human and fungi, which interact with Zn-dependent methionine aminopeptidase type I (MetAP1/Map1p/Fma1). Deletion of the putative metal transferase in Saccharomyces cerevisiae (ZNG1; formerly YNR029c) leads to defective Map1p function and a Zn-deficiency growth defect. Invitro, Zng1p can transfer Zn2+ or Co2+ to apo-Map1p, but unlike characterized copper chaperones, transfer is dependent on GTP hydrolysis. Proteomics reveal mis-regulation of the Zap1p transcription factor regulon because of loss of ZNG1 and Map1p activity, suggesting that Zng1p is required to avoid a compounding effect of Map1p dysfunction on survival during Zn limitation.

Spectrum and characterization of bi-allelic variants in MMAB causing cblB-type methylmalonic aciduria

Pathogenic variants in MMAB cause cblB-type methylmalonic aciduria, an autosomal-recessive disorder of propionate metabolism. MMAB encodes ATP:cobalamin adenosyltransferase, using ATP and cob(I)alamin to create 5’-deoxyadenosylcobalamin (AdoCbl), the cofactor of methylmalonyl-CoA mutase (MMUT). We identified bi-allelic disease-causing variants in MMAB in 97 individuals with cblB-type methylmalonic aciduria, including 33 different and 16 novel variants. Missense changes accounted for the most frequent pathogenic alleles (p.(Arg186Trp), N = 57; p.(Arg191Trp), N = 19); while c.700C > T (p.(Arg234*)) was the most frequently identified truncating variant (N = 14). In fibroblasts from 76 affected individuals, the ratio of propionate incorporation in the presence and absence of hydroxocobalamin (PI ratio) was associated to clinical cobalamin responsiveness and later disease onset. We found p.(Arg234*) to be associated with cobalamin responsiveness in vitro, and clinically with later onset; p.(Arg186Trp) and p.(Arg191Trp) showed no clear cobalamin responsiveness and early onset. Mapping these and novel variants onto the MMAB structure revealed their potential to affect ATP and AdoCbl binding. Follow-up biochemical characterization of recombinant MMAB identified its three active sites to be equivalent for ATP binding, determined by fluorescence spectroscopy (Kd = 21 µM) and isothermal calorimetry (Kd = 14 µM), but function as two non-equivalent AdoCbl binding sites (Kd1 = 0.55 μM; Kd2 = 8.4 μM). Ejection of AdoCbl was activated by ATP (Ka = 24 µM), which was sensitized by the presence of MMUT (Ka = 13 µM). This study expands the landscape of pathogenic MMAB variants, provides association of in vitro and clinical responsiveness, and facilitates insight into MMAB function, enabling better disease understanding.

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 KM(ATP). 31P NMR studies show that binding of the triphosphate product, formed during the adenosylation reaction, is also weakened. However, although the kcat 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.

Cloning and characterization of cobA, one of vitamin B12 biosynthesis pathway genes from Selenomonas ruminantium

Bacterial biosynthesis of vitamin B12 can occur via either aerobic or anaerobic route. While the aerobic pathway has been fully elucidated and understood, less is known about the anaerobic pathway. Selenomonas ruminantium is thought to be the main producer of this vitamin in rumen environment and must use the anaerobic pathway. In our work we found one of the genes of vitamin B12 biosynthetic pathway of S. ruminantium, encoding for the cobalamin adenosyltransferase, enzyme taking part at the last steps of the synthesis process. Deduced amino acid sequence showed the highest similarity to cobalamin adenosyltransferases of other ruminal anaerobic bacteria and that of species Selenomonas. Phylogenetic comparisons of CobA protein sequences of several anaerobic bacteria of Clostridiale order indicate possible horizontal transfer of this gene.

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.

The Crystal Structure of the C-Terminal Domain of the Salmonella enterica PduO Protein: An Old Fold with a New Heme-Binding Mode.

The two-domain protein PduO, involved in 1,2-propanediol utilization in the pathogenic Gram-negative bacterium Salmonella enterica is an ATP:Cob(I)alamin adenosyltransferase, but this is a function of the N-terminal domain alone. The role of its C-terminal domain (PduOC) is, however, unknown. In this study, comparative growth assays with a set of Salmonella mutant strains showed that this domain is necessary for effective in vivo catabolism of 1,2-propanediol. It was also shown that isolated, recombinantly-expressed PduOC binds heme in vivo. The structure of PduOC co-crystallized with heme was solved (1.9 Å resolution) showing an octameric assembly with four heme moieities. The four heme groups are highly solvent-exposed and the heme iron is hexa-coordinated with bis-His ligation by histidines from different monomers. Static light scattering confirmed the octameric assembly in solution, but a mutation of the heme-coordinating histidine caused dissociation into dimers. Isothermal titration calorimetry using the PduOC apoprotein showed strong heme binding (Kd = 1.6 × 10−7 M). Biochemical experiments showed that the absence of the C-terminal domain in PduO did not affect adenosyltransferase activity in vitro. The evidence suggests that PduOC:heme plays an important role in the set of cobalamin transformations required for effective catabolism of 1,2-propanediol. Salmonella PduO is one of the rare proteins which binds the redox-active metabolites heme and cobalamin, and the heme-binding mode of the C-terminal domain differs from that in other members of this protein family.

Diol Dehydratase-Reactivase Is Essential for Recycling of Coenzyme B12 in Diol Dehydratase.

Holoenzymes of adenosylcobalamin-dependent diol and glycerol dehydratases undergo mechanism-based inactivation by glycerol and O2 inactivation in the absence of substrate, which accompanies irreversible cleavage of the coenzyme Co-C bond. The inactivated holodiol dehydratase and the inactive enzyme·cyanocobalamin complex were (re)activated by incubation with NADH, ATP, and Mg(2+) (or Mn(2+)) in crude extracts of Klebsiella oxytoca, suggesting the presence of a reactivating system in the extract. The reducing system with NADH could be replaced by FMNH2. When inactivated holoenzyme or the enzyme·cyanocobalamin complex, a model of inactivated holoenzyme, was incubated with purified recombinant diol dehydratase-reactivase (DD-R) and an ATP:cob(I)alamin adenosyltransferase in the presence of FMNH2, ATP, and Mg(2+), diol dehydratase activity was restored. Among the three adenosyltransferases (PduO, EutT, and CobA) of this bacterium, PduO and CobA were much more efficient for the reactivation than EutT, although PduO showed the lowest adenosyltransfease activity toward free cob(I)alamin. These results suggest that (1) diol dehydratase activity is maintained through coenzyme recycling by a reactivating system for diol dehydratase composed of DD-R, PduO adenosyltransferase, and a reducing system, (2) the releasing factor DD-R is essential for the recycling of adenosycobalamin, a tightly bound, prosthetic group-type coenzyme, and (3) PduO is a specific adenosylating enzyme for the DD reactivation, whereas CobA and EutT exert their effects through free synthesized coenzyme. Although FMNH2 was mainly used as a reductant in this study, a natural reducing system might consist of PduS cobalamin reductase and NADH.

Methylmalonic aciduria cblB type: characterization of two novel mutations and mitochondrial dysfunction studies

Methylmalonic aciduria (MMA) cblB type is caused by mutations in the MMAB gene, which codes for the enzyme adenosine triphosphate (ATP): cobalamin adenosyltransferase (ATR). This study reports differences in the metabolic and disease outcomes of two pairs of siblings with MMA cblB type, respectively harbouring the novel changes p.His183Leu/p.Arg190dup (P1 and P2) and the previously described mutations p.Ile96Thr/p.Ser174fs (P3 and P4). Expression analysis showed p.His183Leu and p.Arg190dup to be destabilizing mutations. Both were associated with reduced ATR stability and a shorter half-life than wild-type ATR. Analysis of several parameters related to oxidative stress and mitochondrial function showed an increase in reactive oxygen species (ROS) content, a decrease in mitochondrial respiration and changes in mitochondria morphology and structure in patient-derived fibroblasts compared to control cells. The impairment in energy production and the presence of oxidative stress and fission of the mitochondrial reticulum suggested mitochondrial dysfunction in cblB patients' fibroblasts. The recovery of mitochondrial function should be a goal in efforts to improve the clinical outcome of MMA cblB type.

A dodecylamine derivative of cyanocobalamin potently inhibits the activities of cobalamin-dependent methylmalonyl-CoA mutase and methionine synthase of Caenorhabditis elegans

In this study, we showed that cyanocobalamin dodecylamine, a ribose 5′-carbamate derivative of cyanocobalamin, was absorbed and accumulated to significant levels by Caenorhabditis elegans and was not further metabolized. The levels of methylmalonic acid and homocysteine, which serve as indicators of cobalamin deficiency, were significantly increased in C. elegans treated with the dodecylamine derivative, indicating severe cobalamin deficiency. Kinetic studies show that the affinity of the cyanocobalamin dodecylamine derivative was greater for two cobalamin-dependent enzymes, methylmalonyl-CoA mutase and methionine synthase, compared with their respective coenzymes, suggesting that the dodecylamine derivative inactivated these enzymes. The dodecylamine derivative did not affect the levels of mRNAs encoding these enzymes or those of other proteins involved in intercellular cobalamin metabolism, including methylmalonyl-CoA mutase (mmcm-1), methylmalonic acidemia cobalamin A complementation group (mmaa-1), methylmalonic aciduria cblC type (cblc-1), and methionine synthase reductase (mtrr-1). In contrast, the level of the mRNAs encoding cob(I)alamin adenosyltransferase (mmab-1) was increased significantly and identical to that of cobalamin-deficient C. elegans. These results indicate that the cyanocobalamin-dodecylamine derivative acts as a potent inhibitor of cobalamin-dependent enzymes and induces severe cobalamin deficiency in C. elegans.

Pharmacological chaperones as a potential therapeutic option in methylmalonic aciduria cblB type

Methylmalonic aciduria (MMA) cblB type is caused by mutations in the MMAB gene. This encodes the enzyme ATP:cob(I)alamin adenosyltransferase (ATR), which converts reduced cob(I)alamin to an active adenosylcobalamin cofactor. We recently reported the presence of destabilizing pathogenic mutations that retain some residual ATR activity. The aim of the present study was to seek pharmacological chaperones as a tailored therapy for stabilizing the ATR protein. High-throughput ligand screening of over 2000 compounds was performed; six were found to enhance the thermal stability of purified recombinant ATR. Further studies using a well-established bacterial system in which the recombinant ATR protein was expressed in the presence of these six compounds, showed them all to increase the stability of the wild-type ATR and the p.Ile96Thr mutant proteins. Compound V (N-{[(4-chlorophenyl)carbamothioyl]amino}-2-phenylacetamide) significantly increased this stability and did not act as an inhibitor of the purified protein. Importantly, compound V increased the activity of ATR in patient-derived fibroblasts harboring the destabilizing p.Ile96Thr mutation in a hemizygous state to within control range. When cobalamin was coadministrated with compound V, mutant ATR activity further improved. Oral administration of low doses of compound V to C57BL/6J mice for 12 days, led to increase in steady-state levels of ATR protein in liver and brain (disease-relevant organs). These results hold promise for the clinical use of pharmacological chaperones in MMA cblB type patients harboring chaperone-responsive mutations.

High resolution melting analysis of the MMAB gene in cblB patients and in those with undiagnosed methylmalonic aciduria

Isolated methylmalonic aciduria (MMA) results either from a defect in the mitochondrial enzyme methylmalonylCoA mutase (MCM), or in the intracellular conversion of vitamin B12 (cobalamin) into its active coenzyme adenosylcobalamin (AdoCbl). Mutations in the MMAB gene affect the function of the enzyme ATP:cob(I)alamin adenosyltransferase (ATR) and the production of AdoCbl. Measurement of MCM function in cultured patient fibroblasts, followed by somatic cell complementation analysis in cases where MCM function is decreased, has classically been used to diagnose the cblB cobalamin disorder. A patient with persistent MMA, who could not be diagnosed using traditional somatic cell studies, was subsequently shown by sequencing in a clinical laboratory to contain two variants in the MMAB gene. This observation brings into question whether somatic cell studies have failed to diagnose other cblB patients with mild cellular phenotypes. A high resolution melting analysis (HRMA) assay was developed for the MMAB gene. It was used to scan 96 reference samples and two cohorts of patients: 42 patients diagnosed with cblB by complementation studies; and 181 patients with undiagnosed MMA. MMAB mutations, including one novel nonsense mutation (c.12 C>A [p.C4X]), were identified in all members of the cblB cohort. Four patients with undiagnosed MMA, including the index case described above, were found to contain variants in the MMAB gene: c.185C>T (p.T62M), c.394T>C (p.C132R), c.398C>T (p.S133F), c.521C>T (p.S174L), c.572G>A (p.R191Q). Only the index case was found to have two variants, suggesting that somatic cell studies diagnose almost all cblB patients.

Clinical and molecular findings in Thai patients with isolated methylmalonic acidemia

Isolated methylmalonic acidemia (MMA) is a genetically heterogeneous organic acid disorder caused by either deficiency of the enzyme methylmalonyl-CoA mutase (MCM), or a defect in the biosynthesis of its cofactor, adenosyl-cobalamin (AdoCbl). Herein, we report and review the genotypes and phenotypes of 14 Thai patients with isolated MMA. Between 1997 and 2011, we identified 6 mut patients, 2 cblA patients, and 6 cblB patients. The mut and cblB patients had relatively severe phenotypes compared to relatively mild phenotypes of the cblA patients. The MUT and MMAB genotypes were also correlated to the severity of the phenotypes. Three mutations in the MUT gene: c.788G>T (p.G263V), c.809_812dupGGGC (p.D272Gfs*2), and c.1426C>T (p.Q476*); one mutation in the MMAA gene: c.292A>G (p.R98G); and three mutations in the MMAB gene: c.682delG (p.A228Pfs*2), c.435delC (p.F145Lfs*69), and c.585-1G>A, have not been previously reported. RT-PCR analysis of a common intron 6 polymorphism (c.520-159C>T) of the MMAB gene revealed that it correlates to deep intronic exonization leading to premature termination of the open reading frame. This could decrease the ATP:cobalamin adenosyltransferase (ATR) activity resulting in abnormal phenotypes if found in a compound heterozygous state with a null mutation. We confirm the genotype–phenotype correlation of isolated MMA in the study population, and identified a new molecular basis of the cblB disorder.

Facilitated Expression and Function Identification of Key Genes Converting Cyanocobalamin to Adenosylcobalamin

Klebsiella pneumoniae degrades glycerol generating 1,3-propanediol(1,3-PD) and 3-hydroxypropionic acid(3-HP) by a pathway dependent on coenzyme B12.Previous studies showed that glycerol could cause the irreversible cleavage of Co— C of coenzyme B12,inactivating glycerol dehydratase(GDHt).For further study on the course of converting inactive vitamin B12(CNCbI) to active coenzyme B12(AdoCbI),ATP:cob(I) alamin adenosyltransferase(ACA) gene btuR and dehydrogenase gene yciK from K.pneumoniae were cloned and a dual-promoter expression plasmid pUC18-tac-btuR-tac-yciK,including gene btuR and yciK,were successfully constructed.Finally,a strain of recombinant bacterium was obtained by transferring the plasmid into Escherichia coli JM109.The results of the improved analytical methods showed that(i) overexpressed proteins had cob(I) alamin adenosyltransferase activity;(ii) the recombinant E.coli JM109 could convert inactive cobalamin,such as vitamin B12,to active coenzyme B12very well.

Loss of Allostery and Coenzyme B12 Delivery by a Pathogenic Mutation in Adenosyltransferase

ATP-dependent cob(I)alamin adenosyltransferase (ATR) is a bifunctional protein: an enzyme that catalyzes the adenosylation of cob(I)alamin and an escort that delivers the product, adenosylcobalamin (AdoCbl or coenzyme B(12)), to methylmalonyl-CoA mutase (MCM), resulting in holoenzyme formation. Failure to assemble holo-MCM leads to methylmalonic aciduria. We have previously demonstrated that only 2 equiv of AdoCbl bind per homotrimer of ATR and that binding of ATP to the vacant active site triggers ejection of 1 equiv of AdoCbl from an adjacent site. In this study, we have mimicked in the Methylobacterium extorquens ATR, a C-terminal truncation mutation, D180X, described in a patient with methylmalonic aciduria, and characterized the associated biochemical penalties. We demonstrate that while k(cat) and K(M)(Cob(I)) for D180X ATR are only modestly decreased (by 3- and 2-fold, respectively), affinity for the product, AdoCbl, is significantly diminished (400-fold), and the negative cooperativity associated with its binding is lost. We also demonstrate that the D180X mutation corrupts ATP-dependent cofactor ejection, which leads to transfer of AdoCbl from wild-type ATR to MCM. These results suggest that the pathogenicity of the corresponding human truncation mutant results from its inability to sequester AdoCbl for direct transfer to MCM. Instead, cofactor release into solution is predicted to reduce the capacity for holo-MCM formation, leading to disease.

Crystal structure of PduO-Type ATP:Cob(I)alamin adenosyltransferase from Bacillus cereus in a complex with ATP

ATP:Cobalamin adenosyltransferases catalyze the transfer a 5′-deoxyadenosyl moiety from ATP to cob(I)alamin in the synthesis of the Co–C bond of coenzyme B12. There are three types of adenosyltransferases, CobA, PduO and EutT. Among these adenosyltransferases, the PduO-type adenosyltransferases is the most widely distributed enzyme. Structural comparisons between apo BcPduO and BcPduO in complex with MgATP revealed that the N-terminal strands of both structures were ordered, which is in contrast with the most previously available PduO-type adenosyltransferase structures. Furthermore, unlike other reported structures, apo BcPduO was bound to additional dioxane molecules causing a side chain conformational change at the Tyr30 residue, which is an important residue that mediates hydrogen bonding with ATP molecules upon binding of cobalamin to the active site. This study provides more structural information into the role of active site residues on substrate binding.

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

Our mechanistic understanding of the conversion of vitamin B(12) into coenzyme B(12) (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.

Functional and structural analysis of five mutations identified in methylmalonic aciduria cbIB type

ATP:cob(I)alamin adenosyltransferase (ATR, E.C.2.5.1.17) converts reduced cob(I)alamin to the adenosylcobalamin cofactor. Mutations in the MMAB gene encoding ATR are responsible for the cblB type methylmalonic aciduria. Here we report the functional analysis of five cblB mutations to determine the underlying molecular basis of the dysfunction. The transcriptional profile along with minigenes analysis revealed that c.584G>A, c.349-1G>C, and c.290G>A affect the splicing process. Wild-type ATR and the p.I96T (c.287T>C) and p.R191W (c.571C>T) mutant proteins were expressed in a prokaryote and a eukaryotic expression systems. The p.I96T protein was enzymatically active with a K(M) for ATP and K(D) for cob(I)alamin similar to wild-type enzyme, but exhibited a 40% reduction in specific activity. Both p.I96T and p.R191W mutant proteins are less stable than the wild-type protein, with increased stability when expressed under permissive folding conditions. Analysis of the oligomeric state of both mutants showed a structural defect for p.I96T and also a significant impact on the amount of recovered mutant protein that was more pronounced for p.R191W that, along with the structural analysis, suggest they might be misfolded. These results could serve as a basis for the implementation of pharmacological therapies aimed at increasing the residual activity of this type of mutations.

Metabolic and Hemodynamic Advantages of an Acetate-Free Citrate Dialysate in a Uremic Case of Congenital Methylmalonic Acidemia

Methylmalonic acidemia (MMA) is an organic acidemia in the class of diseases caused by enzymatic defects in the catabolism of branched-chain amino acids (valine, isoleucine, methionine, and threonine), odd-chain fatty acids, and cholesterol. MMA is an autosomal recessive disorder caused by a deficiency in methylmalonyl–coenzyme A (CoA) mutase (encoded by the MUT gene) or ATP:cob(I)alamin adenosyltransferase activity (encoded by the MMAB gene).1 The causes of MMA are heterogeneous, but the clinical presentation of patients is similar.