Biosynthesis of vitamin B12: the preparative multi-enzyme synthesis of precorrin-3A and 20-methylsirohydrochlorin (a 2,7,20-trimethylisobacteriochlorin)

摘要

pThe iBacillus subtilis/i genes ihemB/i, ihemC/i and ihemD/i, encoding respectively the enzymes porphobilinogen synthase, hydroxymethylbilane synthase and uroporphyrinogen III synthase, have been expressed in iEscherichia coli/i using a single plasmid construct. An enzyme preparation from this source converts 5-aminolaevulinic acid (ALA) preparatively and in high yield into uroporphyrinogen III. The iPseudomonas denitrificans/i genes icobA/i and icobI/i, encoding respectively the enzymes iS/i-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT) and iS/i-adenosyl-L-methionine:precorrin-2 methyltransferase (SPsub2/subMT), were also expressed in iE. coli/i. When SUMT was combined with the coupled-enzyme system that produces uroporphyrinogen III, precorrin-2 was synthesized from ALA, and when SPsub2/subMT was also added the product from the coupling of five enzymes was precorrin-3A. Both of these products are precursors of vitamin Bsub12/sub, and they can be used directly for biosynthetic experiments or isolated as their didehydro octamethyl esters in & 40% overall yield. The enzyme system which produces precorrin-3A is sufficiently stable to allow long incubations on a large scale, affording substantial quantities (15-20 mg) of product./p