| Vitamin B12 (cobalamin, Cbl) is an essential vitamin in humans. Genetic disorders linked to Cbl metabolism have been defined through complementation analysis, and have been assigned to metabolic steps through biochemical analysis. Eight complementation groups have been identified, cblA through cblH. These genetic defects produce functional deficiency in one or both of methionine synthase (MS) or methylmalonyl CoA mutase (MCM), depending on the nature of the block. My quest was to identify missing pieces of the vitamin B12 pathway.; Upon examining prokaryotic operon structures using NCBI's "Cluster of Orthologous Genes" (COG) database, I observed a gene flanking MCM in several genomes and another gene in one genome with similarity to human genes. These studies led to the identification of two genes, MMAA and MMAB, responsible for the cblA and cblB complementation groups, respectively.; Biochemical examination of the MMAA protein was conducted on YgfD (the E. coli ortholog). YgfD was purified, the GTPase activity examined and the interaction with MCM studied. Celis defined the role of YgfD as a lysine-ornithine-arginine (LOA) transporter, while Korotkova demonstrated its role in preventing the inactivation of MCM. While the case for LOA transporter role is severely flawed, the role as a protein preventing inactivation of MCM is in conflict with the information known. My studies suggest that YgfD (and the orthologous MMAA protein) may play a role in a larger complex thereby affecting both MCM activity and AdoCbl levels.; Three patients were examined for mutations in methylmalonyl CoA epimerase (MCEE). Unlike most cblA patients, these three lacked mutations in the MMAA gene, had high AdoCbl levels and were not responsive to vitamin B12 treatment. One patient, WG2278, is homozygous to R49X in MCEE. The family was also found to be homozygous for R49X. A knockdown of the epimerase expression, using siRNA, revealed that 14C-propionate uptake was affected.; A region of chromosome 1p34 was identified by Atkinson to be critical to cblC patients. Sixteen candidate genes belonging to that region were tested for mutation in cblC cell lines. Several single nucleotide polymorphisms were identified in these genes, including, a two base pair (AT) deletion. No gene was identified as the gene responsible for the cblC disorder.; To identify and test additional genes in vitamin B12 metabolism, I developed a method of "genome subtraction" in which all the genes found in microbial vitamin B12 users are subtracted from non-users leaving only vitamin B12 genes. (Abstract shortened by UMI.)... |
