Biosynthesis of vitamin B12: the preparative multi-enzyme 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 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.
University Chemical Laboratory, University of Cambridge, U.K.
N P
NP
Stamford
J
J
Crouzet
B
B
Cameron
A I
AI
Alanine
A R
AR
Pitt
A A
AA
Yeliseev
A R
AR
Battersby
D001412:Bacillus subtilis / Q000201:enzymology; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D011163:Hydroxymethylbilane Synthase / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D000623:Porphobilinogen Synthase / Q000235:genetics / Q000378:metabolism; D014576:Uroporphyrinogen III Synthetase / Q000235:genetics / Q000378:metabolism; D014577:Uroporphyrinogens / Q000378:metabolism; D014578:Uroporphyrins / Q000096:biosynthesis*; D014805:Vitamin B 12 / Q000096:biosynthesis*
C097627:20-methylsirohydrochlorin; D014577:Uroporphyrinogens; D014578:Uroporphyrins; C078447:precorrin 3; D011163:Hydroxymethylbilane Synthase; D000623:Porphobilinogen Synthase; D014576:Uroporphyrinogen III Synthetase; D014805:Vitamin B 12