Cloning, sequencing and expression of the gene encoding the coenzyme B12‐dependent 2‐methyleneglutarate mutase from Clostridium barkeri in Escherichia coli

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.