The antipernicious anemia factor, vitamin B-12 (cyanocobalamin) is a member of a family of complex tetrapyrroles, the corrinoids. The corrinoids are ancient in the evolutionary sense, apparently functionally predating the porphyrins. They are chemically unique, being the only known natural products possessing alkyl-cobalt covalent bonds. Complete corrinoid biosynthesis, the nearly exclusive purview of certain bacterial genera, is as yet not completely understood. The early steps in corrinoid biosynthesis, formation of the ring, related methylations, and insertion of the cobalt atom, as well as the final stages of cobalamin formation, i.e. attachment of the nucleotide loop, have been successfully or are currently being studied in detail in several laboratories. In contrast, detailed investigations of the intermediate steps, amidation of and isopropanolamine attachment to the corrin ring, have been reported in the literature only recently. In addition, the recent discovery that certain putative cobalamin precursors can function as reductive catalysts for organo-halide dehalogenations suggests incomplete corrinoids may be of key importance in prokaryotic metabolic detoxification processes. Thus, the purposes of the proposed research are to 1) investigate the enzymatic details of the intermediate cobamide biosynthetic steps, and 2) study the chemical and physical properties of the """"""""incomplete"""""""" corrinoids which may take part in both the biosynthetic pathway and be important in dehalogenation/detoxification metabolism. Successful pursuit of these goals will result in elucidation of unique microbial biosynthetic and metabolic pathways, an understanding of which will lead to further clarification of corrin function in both bacterial and mammalian systems. The specific project design is as follows: 1) structural characterization of prepared """"""""incomplete"""""""" corrinoid substrates by degradation of vitamin B-12, 2) modification of """"""""incomplete"""""""" corrinoid substrates to prepare unique corrinoids for further chemical study and/or use in enzyme assay, 3) development of reproducible methods for assay of enzyme activity and of corrinoids as nonenzymatic dehalogenation catalysts, 4) development of techniques for preparation and purification of biosynthetically active extracts from corrinoid-producer organisms, 5) utilization of purified enzyme preparations to study unresolved biosynthetic details of cobalamin biosynthesis, 6) use of bacterial corrinoid extracts and purified incomplete corrinoids to study the structure/function characteristics of the reductive dehalogenation reactions.
