Cloning, sequencing and expression of the gene encoding the coenzyme B12-dependent 2-methyleneglutarate mutase from Clostridium barkeri in Escherichia coli.
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.
Beatrix
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B
Laboratorium für Mikrobiologie des Fachbereichs Biologie, Philipps-Universität Marburg, Germany.
Zelder
O
O
Linder
D
D
Buckel
W
W
D000595:Amino Acid Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D003013:Clostridium / Q000235:genetics*; D003038:Cobamides / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D015723:Gene Library; D005798:Genes, Bacterial; D019751:Intramolecular Transferases; D007535:Isomerases / Q000235:genetics*; D008027:Light; D008969:Molecular Sequence Data; D017421:Sequence Analysis; D017385:Sequence Homology
D003038:Cobamides; D007535:Isomerases; D019751:Intramolecular Transferases; C047997:2-methyleneglutarate mutase; C000913:cobamamide