Cobamide structure depends on both lower ligand availability and CobT substrate specificity.

Cobamides are members of the vitamin B12 family of cofactors that function in a variety of metabolic processes and are synthesized only by prokaryotes. Cobamides produced by different organisms vary in the structure of the lower axial ligand. Here we explore the molecular factors that control specificity in the incorporation of lower ligand bases into cobamides. We find that the cobT gene product, which activates lower ligand bases for attachment, limits the range of lower ligand bases that can be incorporated by bacteria. Furthermore, we demonstrate that the substrate specificity of CobT can be predictably altered by changing two active site residues. These results demonstrate that sequence variations in cobT homologs contribute to cobamide structural diversity. This analysis could open new routes to engineering specific cobamide production and understanding cobamide-dependent processes.