Functional characterization and mutation analysis of human ATP:Cob(I)alamin adenosyltransferase.
ATP:cob(I)alamin adenosyltransferase catalyzes the final step in the conversion of vitamin B 12 into the active coenzyme, adenosylcobalamin. Inherited defects in the gene for the human adenosyltransferase (hATR) result in methylmalonyl aciduria (MMA), a rare but life-threatening illness. In this study, we conducted a random mutagenesis of the hATR coding sequence. An ATR-deficient strain of Salmonella was used as a surrogate host to screen for mutations that impaired hATR activity in vivo. Fifty-seven missense mutations were isolated. These mapped to 30 positions of the hATR, 25 of which had not previously been shown to impair enzyme activity. Kinetic analysis and in vivo tests for enzyme activity were performed on the hATR variants, and mutations were mapped onto a hATR structural model. These studies functionally defined the hATR active site and tentatively implicated three amino acid residues in facilitating the reduction of cob(II)alamin to cob(I)alamin which is a prerequisite to adenosylation.
Fan
Chenguang
C
Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA.
Bobik
Thomas A
TA
D000255:Adenosine Triphosphate / Q000378:metabolism*; D019883:Alkyl and Aryl Transferases / Q000235:genetics* / Q000378:metabolism*; D015153:Blotting, Western; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D008958:Models, Molecular; D016296:Mutagenesis; D020125:Mutation, Missense; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013056:Spectrophotometry, Ultraviolet; D014805:Vitamin B 12 / Q000494:pharmacology
D000255:Adenosine Triphosphate; D019883:Alkyl and Aryl Transferases; C019920:cob(I)alamin adenosyltransferase; D014805:Vitamin B 12