| In humans, the B12 coenzymes, adenosylcobalamin and methylcobalamin, are required cofactors for propionate metabolism and methionine biosynthesis, respectively. Humans are incapable of de novo synthesis of the B12 coenzymes and require complex precursors such as vitamin Bit in their diet. The metabolism of vitamin B12 to adenosylcobalamin requires two successive reductions and an adenosylation reaction; however, many aspects of the genetics and biochemistry of this process have not been elucidated. This dissertation focuses on the isolation and characterization of the human genes and enzymes involved in the formation of adenosylcobalamin from vitamin B12. Deficiencies in this process result in methylmalonic aciduria, a rare disorder that is often fatal in newborns.; Because of the sophisticated genetic methods that are available, Salmonella enterica was used as a model system. By complementation of an S. enterica mutant, a bovine adenosyltransferase cDNAs was isolated and subsequent sequence similarity searches identified a homologous human cDNA. The bovine and human cDNAs were independently cloned, expressed in Escherichia coli, and the encoded proteins were shown to have adenosyltransferase activities similar to previously studied bacterial adenosyltransferases. Additional studies, using Western blots, showed that adenosyltransferase expression was altered in cell lines derived from patients with cblB methylmalonic aciduria when compared to cell lines from normal individuals.; Two common human adenosyltransferase polymorphic variants were expressed in E. coli, purified, and biochemically characterized. Both had high specificity for ATP and comparable Km and Vmax values. An adenosyltransferase-linked cob(II)alamin reductase assay was developed and used to show that the human methionine synthase reductase functions as a cob(II)alamin reductase for adenosylcobalamin formation. A linked assay was also performed in the presence of a cob(I)alamin trapping agent, and results from these experiments suggested that human adenosyltransferase and methionine synthase reductase interact. Importantly, results from this work provide information that will allow improvements in diagnosis and treatment of methylmalonic aciduria, a devastating childhood disorder. |
