Biosynthesis of Vitamin B12: the Preparative Multi-enzyme Synthesis of Precorrin-3A and 20-methylsirohydrochlorin (a 2,7,20-trimethylisobacteriochlorin)

Overview

Journal Biochem J

Specialty Biochemistry

Date 1996 Jan 1

PMID 8546704

Authors

N P Stamford

J Crouzet

B Cameron

A I Alanine

A R Pitt

A A Yeliseev

A R BATTERSBY

Affiliations

Soon will be listed here.

Abstract

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.

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