[Microbe-inspired system enzymology of vitamin B12 metabolism].

Vitamin B12 is produced only by prokaryotes and utilized by animals as an essential micronutrient. Genetic complementation analysis of cell lines from patients indicated that at least eight gene products are involved in intracellular B12 metabolism and utilization. We have investigated bacterial adenosylcobalamin-dependent enzymes and elucidated their structure-based fine mechanisms. They tend to undergo mechanism-based inactivation during catalysis, because they use highly reactive radicals for catalyzing chemically difficult reactions. We have discovered molecular chaperone-like reactivating factors for these enzymes that release a damaged cofactor forming apoenzyme. Methylcobalamin-dependent methionine synthase also undergoes inactivation, because it utilizes cob (I) alamin, a super nucleophile, for catalysis. Methionine synthase reductase is a reactivating partner for this enzyme. Recent studies suggested that activity-maintaining systems for B12 enzymes are present in animal cells as well, and thus hints for designing therapeutic agents for B12-related metabolic disorders might be obtained from the investigations of microbial B12 metabolism.

Long-term rescue of a lethal murine model of methylmalonic acidemia using adeno-associated viral gene therapy.

Methylmalonic acidemia (MMA) is an organic acidemia caused by deficient activity of the mitochondrial enzyme methylmalonyl-CoA mutase (MUT). This disorder is associated with lethal metabolic instability and carries a poor prognosis for long-term survival. A murine model of MMA that replicates a severe clinical phenotype was used to examine the efficacy of recombinant adeno-associated virus (rAAV) serotype 8 gene therapy as a treatment for MMA. Lifespan extension, body weight, circulating metabolites, transgene expression, and whole animal propionate oxidation were examined as outcome parameters after gene therapy. One-hundred percent of the untreated Mut(-/-) mice (n = 58) died by day of life (DOL) 72, whereas >95% of the adeno-associated virus-treated Mut(-/-) mice (n = 27) have survived for > or = 1 year. Despite a gradual loss of transgene expression and elevated circulating metabolites in the treated Mut(-/-) mice, the animals are indistinguishable from unaffected control littermates in size and activity levels. These experiments provide the first definitive evidence that gene therapy will have clinical utility in the treatment of MMA and support the development of gene therapy for other organic acidemias.

Propionyl-CoA and adenosylcobalamin metabolism in Caenorhabditis elegans: evidence for a role of methylmalonyl-CoA epimerase in intermediary metabolism.

We have utilized Caenorhabditis elegans to study human methylmalonic acidemia. Using bioinformatics, a full complement of mammalian homologues for the conversion of propionyl-CoA to succinyl-CoA in the genome of C. elegans, including propionyl-CoA carboxylase subunits A and B (pcca-1, pccb-1), methylmalonic acidemia cobalamin A complementation group (mmaa-1), co(I)balamin adenosyltransferase (mmab-1), MMACHC (cblc-1), methylmalonyl-CoA epimerase (mce-1) and methylmalonyl-CoA mutase (mmcm-1) were identified. To verify predictions that the entire intracellular adenosylcobalamin metabolic pathway existed and was functional, the kinetic properties of the C. elegans mmcm-1 were examined. RNA interference against mmcm-1, mmab-1, mmaa-1 in the presence of propionic acid revealed a chemical phenotype of increased methylmalonic acid; deletion mutants of mmcm-1, mmab-1 and mce-1 displayed reduced 1-[(14)C]-propionate incorporation into macromolecules. The mutants produced increased amounts of methylmalonic acid in the culture medium, proving that a functional block in the pathway caused metabolite accumulation. Lentiviral delivery of the C. elegans mmcm-1 into fibroblasts derived from a patient with mut(o) class methylmalonic acidemia could partially restore propionate flux. The C. elegans mce-1 deletion mutant demonstrates for the first time that a lesion at the epimerase step of methylmalonyl-CoA metabolism can functionally impair flux through the methylmalonyl-CoA mutase pathway and suggests that malfunction of MCEE may cause methylmalonic acidemia in humans. The C. elegans system we describe represents the first lower metazoan model organism of mammalian propionate spectrum disorders and demonstrates that mass spectrometry can be employed to study a small molecule chemical phenotype in C. elegans RNAi and deletion mutants.

N2O and urine methylmalonic acid in man.

The excretion of methylmalonic acid was measured and deoxyuridine suppression test (dU-test) was performed in 5 patients before and after 24 h of ventilation with nitrous oxide. The mean urinary 2-methylmalonic acid was increased from 90 to 320 mumol/24 h and the dU-tests became abnormal. These findings indicate that the 2 vitamin B-12 dependent enzymes, methylmalonyl-CoA-mutase and methione synthetase, are depressed by nitrous oxide.