Biosynthesis of vitamin B 12: the multi-enzyme synthesis of precorrin-4 and factor IV

Abstract

Background: In order to study the biosynthesis of vitamin B 12, it is necessary to produce various intermediates along the biosynthetic pathway by enzymic methods. Recently, information on the organisation of the biosynthetic pathway has permitted the selection of the set of enzymes needed to biosynthesise any specific identified intermediate. The aim of the present work was to use recombinant enzymes in reconstituted multi-enzyme systems to biosynthesise particular intermediates. Results: The products of the cobG and cobJ genes from Pseudomonas denitrificans were expressed heterologously in Escherichia coli to afford good levels of activity of the corresponding enzymes, CobG and CobJ. Aerobic incubation of precorrin-3A with the CobG enzyme alone yielded precorrin-3B. When CobJ and S-adenosyl- l-methionine were included in the incubation, the product was precorrin-4. Both precorrin-3B and precorrin-4 are known precursors of vitamin B 12 and their availability has allowed new mechanistic studies of enzymic transformations. Conclusions: Our results show that the expression of the CobG and CobJ enzymes has been successful, thus facilitating the biosynthesis of two precursors of vitamin B 12. This lays the foundation for the structure determination of CobG and CobJ as well as future enzymic experiments focusing on later steps of vitamin B 12 biosynthesis.