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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January 1996
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Research Article|
January 01 1996
Biosynthesis of vitamin B12: the preparative multi-enzyme synthesis of precorrin-3A and 20-methylsirohydrochlorin (a 2,7,20-trimethylisobacteriochlorin) Available to Purchase
N. J. Patrick STAMFORD;
N. J. Patrick STAMFORD
*University Chemical Laboratory, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K.
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Joël CROUZET;
Joël CROUZET
†Department Analyse, Centre de Recherché de Alfortville, Rhône-Poulenc Rorer, BP14, F-94403, Vitry-sur-Seine Cedex, France
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Béatrice CAMERON;
Béatrice CAMERON
†Department Analyse, Centre de Recherché de Alfortville, Rhône-Poulenc Rorer, BP14, F-94403, Vitry-sur-Seine Cedex, France
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Alex I. D. ALANINE;
Alex I. D. ALANINE
*University Chemical Laboratory, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K.
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Andrew R. PITT;
Andrew R. PITT
*University Chemical Laboratory, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K.
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Alexei A. YELISEEV;
Alexei A. YELISEEV
*University Chemical Laboratory, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K.
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Alan R. BATTERSBY
Alan R. BATTERSBY
‡
*University Chemical Laboratory, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, U.K.
‡To whom correspondence should be addressed.
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Publisher: Portland Press Ltd
Received:
April 24 1995
Revision Received:
August 07 1995
Accepted:
August 25 1995
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1996
1996
Biochem J (1996) 313 (1): 335–342.
Article history
Received:
April 24 1995
Revision Received:
August 07 1995
Accepted:
August 25 1995
Citation
N. J. Patrick STAMFORD, Joël CROUZET, Béatrice CAMERON, Alex I. D. ALANINE, Andrew R. PITT, Alexei A. YELISEEV, Alan R. BATTERSBY; Biosynthesis of vitamin B12: the preparative multi-enzyme synthesis of precorrin-3A and 20-methylsirohydrochlorin (a 2,7,20-trimethylisobacteriochlorin). Biochem J 1 January 1996; 313 (1): 335–342. doi: https://doi.org/10.1042/bj3130335
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