Genetically Engineered Synthesis and Structural Characterization of Cobalt−Precorrin 5A and −5B, Two New Intermediates on the Anaerobic Pathway to Vitamin B₁₂:  Definition of the Roles of the CbiF and CbiG Enzymes

Two new cobalt corrinoid intermediates, cobalt−precorrin 5A and cobalt−precorrin 5B, have been synthesized with the aid of overexpressed enzymes of the vitamin B₁₂ pathway of Salmonella enterica serovar typhimurium. These compounds were made in several regioselectively ¹³C-labeled forms, and their structures have been established by multidimensional NMR spectroscopy. The addition of CbiF to the enzymes known to synthesize cobalt−precorrin 4 resulted in the formation of cobalt−precorrin 5A, and the inclusion of CbiG with CbiF produced cobalt−precorrin 5B, which has allowed us to define the role of these enzymes in the anaerobic biosynthetic pathway. CbiF is the C-11 methylase, and CbiG, an enzyme which shows homology with CobE of the aerobic pathway, is the gene product responsible for the opening of the ring A δ-lactone and extrusion of the “C₂” unit. The discovery of these long-sought intermediates paves the way for defining the final stages of the anaerobic pathway. It is of considerable evolutionary interest that nature uses two distinct pathways to vitamin B₁₂, both conserved over several billion years and featuring completely different mechanisms for ring-contraction of the porphyrinoid to the corrinoid ring system. Thus the aerobic pathway utilizes molecular oxygen to trigger the events at C-20 leading to contraction and expulsion of the “C₂” unit as acetic acid from a metal-free intermediate, whereas the anaerobic route features internal delivery of oxygen from a carboxylic acid terminus to C-20 followed by extrusion of the “C₂” unit as acetaldehyde, using cobalt complexes as substrates.