The transfer of a methyl group from C-I 1 to C-12 in vitamin BI2 biosynthesis is shown, by a crossover experiment involving [ I ,I 1,17-(CD3)3]-labelled precursors, to occur in an intramolecular fashion; it is found that in Pseudomonas denitrificans, unlike Propionibacterium shermanii, no exchange of the ring A acetate oxygen atoms occurs during formation of the corrin ring.

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Our results show that the expression of the CobG and CobJ enzymes has been successful, thus facilitating the biosynthesis of two precursors of vitamin B12. This lays the foundation for the structure determination of CobG and CobJ as well as future enzymic experiments focusing on later steps of vitamin B12 biosynthesis.

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I ,I 0,20-W3]Uro'gen Ill is unambiguously synthesised for enzymic conversion into precorrin-4 isolated after aerial oxidation as two epimers of Factor IV, the 13C NMR spectra of which rigorously confirm the presence of a C-I acetyl group in precorrin-4; attachment of the fourth methyl group at C-17 of precorrin-4 is also confirmed by related 1%-labelling experiments.

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The Bacillus subtilis genes hemB, hemC and hemD, encoding respectively the enzymes porphobilinogen synthase, hydroxymethylbilane synthase and uroporphyrinogen III synthase, have been expressed in Escherichia coli 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 Pseudomonas denitrificans genes cobA and cobI, encoding respectively the enzymes S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT) and S-adenosyl-L-methionine:precorrin-2 methyltransferase (SP2MT), were also expressed in E. coli. When SUMT was combined with the coupled-enzyme system that produces uroporphyrinogen III, precorrin-2 was synthesized from ALA, and when SP2MT was also added the product from the coupling of five enzymes was precorrin-3A. Both of these products are precursors of vitamin B12, 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.