BIOSYNTHESIS OF VITAMIN-B-12 - THE PREPARATIVE MULTIENZYME SYNTHESIS OF PRECORRIN-3A AND 20-METHYLSIROHYDROCHLORIN (A 2,7,20-TRIMETHYLISOBACTERIOCHLORIN)

The Bacillus subtilis genes hemB, hemC and hemD, encoding respectively the enzymes porphobilinogen synthase, hydroxymethylbilane synthase an d uroporphyrinogen III synthase, have been expressed in Escherichia co li using a single plasmid construct. An enzyme preparation from this s ource converts 5-aminolaevulinic acid (ALA) preparatively and in high yield into uroporphyrinogen III. The Pseudomonas denitrificans genes c obA and cobI, encoding respectively the enzymes S-adenosyl-L-methionin e:uroporphyrinogen III methyltransferase (SUMT) and S-adenosyl-L-methi onine:precorrin-2 methyltransferase (SP(2)MT), were also expressed in E. coli. When SUMT was combined with the coupled-enzyme system that pr oduces uroporphyrinogen III, precorrin-2 was synthesized from ALA, and when SP,MT was also added the product from the coupling of five enzym es was precorrin-3A. Both of these products are precursors of vitamin B-12, and they can be used directly for biosynthetic experiments or is olated as their didehydro octamethyl esters in > 40% overall yield. Th e enzyme system which produces precorrin-3A is sufficiently stable to allow long incubations on a large scale, affording substantial quantit ies (15-20 mg) of product.