Vitamin B12 is nature's most magnificent cofactor and represents the largest of the B-type vitamins. It contains a corrin ring system with a central cobalt ion octahedrally coordinated by the four pyrrole nitrogens of the corrin and two axial ligands. The overall goal of this proposal is to understand, on a detailed molecular level, the structural/functional aspects of the enzymes involved in assembling the axial ligands of coenzyme B12 in Salmonella typhimurium. This process requires at least seven enzymes to add the 5'-deoxyadenosyl moiety to the corrin ring, synthesize the """"""""nucleotide loop"""""""" and assemble the lower ligand of cobalamin. A combination of structural and biochemical approaches will be utilized to provide a detailed description of these enzymatic systems. The immediate aims are to define the structural mechanism for how ATP:corrinoid adenosyl transferase (CobA) catalyzes the formation of a cobalt-carbon bond and how kinase/adenosylcobinamide phosphate guanylytransferase (CobU) activates adenosyl cobinamide prior to assembly of the lower ligand. In parallel it is planned to define the structural mechanism of the phosphoribosyltransferase (CobT) responsible for biosynthesis of the lower ligand. The experimental systems necessary to accomplish these goals are well established. The longer term goal of this proposal is to crystallize and then determine the structures of the four other enzymes involved in the final stages of cobalamin biosynthesis, CobD, CbiB, CobC and CobS, to understand the complete structural framework for assembling the lower ligand of this complex coenzyme. Together this study will reveal the structural relationships and potential for biochemical coupling between the enzymes that form the biosynthetic pathway for cobalamin. It will also provide insight into their underlying chemical mechanisms. In addition this study will clarify those factors that influence corrinoid recognition and utilization which will in turn provide a broader framework for understanding vitamin B12 dependent enzymes.
