Biosynthesis of vitamin B12: the multi-enzyme synthesis of precorrin-4 and factor IV.
BACKGROUND
In order to study the biosynthesis of vitamin B12, it is necessary to produce various intermediates along the biosynthetic pathway by enzymic methods. Recently, information on the organisation of the biosynthetic pathway has permitted the selection of the set of enzymes needed to biosynthesise any specific identified intermediate. The aim of the present work was to use recombinant enzymes in reconstituted multi-enzyme systems to biosynthesise particular intermediates.


RESULTS
The products of the cobG and cobJ genes from Pseudomonas denitrificans were expressed heterologously in Escherichia coli to afford good levels of activity of the corresponding enzymes, CobG and CobJ. Aerobic incubation of precorrin-3A with the CobG enzyme alone yielded precorrin-3B. When CobJ and S-adenosyl-L-methionine were included in the incubation, the product was precorrin-4. Both precorrin 3B and precorrin-4 are known precursors of vitamin B12 and their availability has allowed new mechanistic studies of enzymic transformations.


CONCLUSIONS
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.
Stamford
N P
NP
University Chemical Laboratory, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK.
Duggan
S
S
Li
Y
Y
Alanine
A I
AI
Crouzet
J
J
Battersby
A R
AR
D001426:Bacterial Proteins; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D015394:Molecular Structure; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011549:Pseudomonas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism; D014578:Uroporphyrins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D014805:Vitamin B 12 / Q000096:biosynthesis*
D001426:Bacterial Proteins; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D014578:Uroporphyrins; C107384:precorrin 4; C078447:precorrin 3; D010105:Oxygenases; C083406:CobG protein, Pseudomonas denitrificans; C083407:CobJ protein, Pseudomonas denitrificans; D008780:Methyltransferases; D014805:Vitamin B 12