Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
The B12 coenzymes are required cofactors for at least 15 different enzymes that are widely distributed in nature and are essential for human health. Characterizing enzymes in each step of vitamin B12 metabolism in humans will provide important information for human B12-related disease diagnosis and therapy. Human ATP:cob(I)alamin adenosyltransferase (hATR) catalyzes the final step in the conversion of vitamin B12 into the active coenzyme, adenosylcobalamin. Inherited defects in this gene result in a rare but life-threatening disease, methylmalonyl aciduria. We conducted a random mutagenesis of the hATR coding sequence. Fifty-seven missense mutations were isolated mapping to 30 positions, 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.
Human cannot synthesize coenzyme B12 de novo but assimilate B12 complex precursors, which are only from certain prokaryotes. The biosynthesis of B12 has been studied extensively in Salmonella enterica, however, the function of PduX gene involved in B12-dependent propanediol degradation was still unknown. In vitro test including 31P NMR spectroscopy and enzyme assays established that purified PduX-His6 catalyzed the conversion of L-threonine and ATP to L-threonine-O-3-phosphate and ADP. A series of in vivo growth studies showed that PduX is an L-threonine kinase used for AdoCbl synthesis. The PduX enzyme belongs to GHMP kinase family by sequence similarity. Kinetic analysis indicated an ordered ternary complex mechanism in which ATP is the first substrate to bind. The lack of obvious solvent pH and isotope effects indicated that proton transfer is unlikely to be involved directly in the rate determining step of catalysis. Based on a multiple sequence alignment of PduX homologues and other GHMP family members, 14 PduX variant were constructed by site-directed mutagenesis. Comparison of the Circular Dichroism spectra and kinetic properties of the PduX variants to those of the wild-type enzyme functionally defined the L-threonine kinase active site.
Fan, Chenguang, "Biochemical and mutational analysis of coenzyme B12 biosynthesis" (2009). Graduate Theses and Dissertations. 11247.