Evidence that the CysG protein catalyzes the first reaction specific to B-12 synthesis in Salmonella typhimurium, insertion of cobalt

The cysG gene of Salmonella typhimurium is involved in synthesis of both cobalamin (B-12) and siroheme (a cofactor required for SO32- and NO22- reductases). The failure to reduce SO32- leads to cysteine auxotrophy, for which the enzyme is named, Although Escherichia coli does not synthesize B-12 de novo, it possesses a very similar CysG enzyme which has been shown to catalyze two methylations (uroporphyrinogen III to precorrin-2), ring oxidation (precorrin-2 to factor II), and iron insertion (factor II to siroheme). In S. typhimurium, precorrin-2 is a precursor of both siroheme and B-12. All previously known Salmonella cysG mutants are defective in the synthesis of both siroheme and cobalamin. We describe two new classes of cysG mutants that cannot synthesize B-12 but still make siroheme. For class I mutants, exogenous cobalt corrects the B-12 defect but inhibits ability to make siroheme; B-12 synthesis is inhibited by added iron, Class II mutants are unaffected by exogenous cobalt, but their B-12 defect is corrected by derepression of the B-12 biosynthetic genes (cob). We propose that all mutants are defective in insertion of cobalt into factor II and that the Salmonella CysG enzyme normally catalyzes this insertion-the first reaction dedicated to cobalamin synthesis. Although E. coli does not make B its CysG enzyme has been shown in vitro to insert cobalt into factor II and may have evolved to support B-12 synthesis in some ancestor common to Salmonella species and E. coli.