BIOSYNTHESIS OF VITAMIN-B-12 - THE MULTIENZYME SYNTHESIS OF PRECORRIN-4 AND FACTOR-IV

Background: In order to study the biosynthesis of vitamin B-12, it is necessary to produce various intermediates along the biosynthetic path way by enzymic methods. Recently, information on the organisation of t he biosynthetic pathway has permitted the selection of the set of enzy mes needed to biosynthesise any specific identified intermediate. The aim of the present work was to use recombinant enzymes in reconstitute d multi-enzyme systems to biosynthesise particular intermediates. Resu lts: The products of the cobG and cobJ genes from Pseudomonas denitrif icans were expressed heterologously in Escherichia coli to afford good levels of activity of the corresponding enzymes, CobG and CobJ. Aerob ic incubation of precorrin-3A with the CobG enzyme alone yielded preco rrin-3B. When CobJ and S-adenosyl-L-methionine were included in the in cubation, the product was precorrin-4. Both precorrin-3B and precorrin -4 are known precursors of vitamin B-12 and their availability has all owed 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 determinat ion of CobG and CobJ as well as future enzymic experiments focusing on later steps of vitamin B-12 biosynthesis.