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

Abstract

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 racemase 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.

Adenoviral-mediated correction of methylmalonyl-CoA mutase deficiency in murine fibroblasts and human hepatocytes

“…Mut and wild-type murine embryonic fibroblasts have been described[24,25] and were isolated after a timed mating between mice carrying a targeted deletion of the Mut gene.…”

“…A murine methylmalonyl-CoA mutase cDNA that contained a consensus Kozak sequence and minimal untranslated regions was isolated from C57/BL6 liver RNA after RT-PCR, sequenced and tested for enzymatic activity after expression in yeast using the succinate-thiokinase linked assay[25]. The gene was then cloned as an EcoRI fragment into the polylinker of pShuttle between the CMV promoter and the Sv40 polyadenylation signal (ViraQuest Inc., North Liberty, IA).…”

Structural and mechanistic comparisons of the metal-binding members of the vicinal oxygen chelate (VOC) superfamily

“…MMCE catalyzes the conversion of (2R)-methylmalonyl-CoA to (2S)-methylmalonyl-CoA, the substrate for the B 12 -dependent methylmalonyl-CoA mutase. MMCE is widely found and essential in the breakdown of odd-chain fatty acids and branched amino acids [33][34][35]. The Propionibacteriumshermanii enzyme is activated to the greatest extent by Co 2+ , but also by Ni 2+ , Mn 2+ , and Zn 2+ , and the rat liver enzyme by Fe 2+ , Mn 2+ , and Co 2+ [36,37].…”

Associations between gene polymorphisms in two crucial metabolic pathways and growth traits in pigs

“…In addition, we selected three genes known to encode propionyl Coenzyme A (PCCB), methylmalonyl CoA epimerase (MCEE), and methylmalonyl CoA mutase (MUT) respectively, which are involved in the essential process of conversion of propionyl-CoA to succinylCoA. A lesion at the epimerase step of methylmalonyl-CoA metabolism can cause malfunction of MCEE, which may cause methylmalonicacidemia in humans [6]. The last two candidate genes chosen in the propanoate metabolic pathway were ACSS1 and ACSS2, which are known to be highly expressed in the livers of mice to produce lipogenic enzymes that incorporate acetate into lipids [15,16].…”

“…The regulatory mechanisms of the propanoate and fatty acid metabolic pathways have not been clarified. Reports have indicated that genes within the two essential pathways are directly involved with anabolism and catabolism, and they can therefore be speculated to have an effect on the growth of an organism [6][7][8].…”