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- Title
Cloning, sequencing and expression of the gene encoding the coenzyme B12-dependent 2-methyleneglutarate mutase from Clostridium barkeri in Escherichia coli.
- Authors
Beatrix, B; Zelder, O; Linder, D; Buckel, W
- Abstract
The coenzyme B12 (adenosylcobalamin)-dependent 2-methyleneglutarate mutase catalyses the carbon skeleton rearrangement of 2-methyleneglutarate to (R)-3-methylitaconate in the fermentation of nicotinic acid by the strict anaerobic bacterium Clostridium barkeri. (a) The mgm gene encoding 2-methyleneglutarate mutase was cloned and its nucleotide sequence was determined. The deduced amino acid sequence revealed a 66.8-kDa protein of 614 amino acids. It shows significant similarity in its C-terminal part to that of other cobamide-dependent enzymes. Probably, this is the coenzyme-binding region. (b) The mgm gene from C. barkeri was expressed in Escherichia coli as was shown by SDS/PAGE and Western-blot analysis with rabbit antiserum directed against the native mutase. (c) Cell-free extracts from E. coli carrying the mgm gene showed 2-methyleneglutarate mutase activity that was strictly dependent on the addition of coenzyme B12. Experiments are presented which suggest that the expression product is an apoenzyme.
- Publication
European journal of biochemistry, 1994, Vol 221, Issue 1, p101
- ISSN
0014-2956
- Publication type
Journal Article
- DOI
10.1111/j.1432-1033.1994.tb18718.x