Cloning, sequencing and expression in Escherichia coli of the gene encoding component S of the coenzyme B12-dependent glutamate mutase from Clostridium cochlearium.
Adenosylcobalamin (coenzyme B12) dependent glutamate mutase catalyzes the carbon skeleton rearrangement of (S)-glutamate to (2S,3S)-methylaspartate. This is the first step of the fermentation of glutamate by the strict anaerobic bacterium Clostridium cochlearium. The enzyme consists of the two protein components E and S. The gene encoding component S (glmS) was cloned in Escherichia coli and its nucleotide sequence was determined. The nucleotide sequence and the deduced amino acid sequence showed very strong identities to the sequence of the glmS (also called mutS) gene (80%) and to component S (82%) from the related C. tetanomorphum, respectively. Cell-free extracts of E. coli carrying the glmS gene showed glutamate mutase activity which was strictly dependent on the addition of coenzyme B12 and component E purified from C. cochlearium. Enzyme activity of the recombinant protein was achieved up to 2200 nkat/g wet cells which is due to a ten-fold overexpression compared with the activities determined in cell-free extracts of C. cochlearium. This is the first report of overexpression of an active component of glutamate mutase. A rapid purification procedure consisting only of ammonium sulfate precipitation and a gel filtration step was developed to obtain large amounts of pure component S in a short time.
Zelder
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Laboratorium für Mikrobiologie, Fachbereich Biologie, Philips-Universität, Marburg, FRG.
Beatrix
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Buckel
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D000591:Amino Acid Isomerases / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology / Q000235:genetics*; D004269:DNA, Bacterial / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D019751:Intramolecular Transferases; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D017421:Sequence Analysis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)
D000591:Amino Acid Isomerases; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D019751:Intramolecular Transferases; D011993:Recombinant Fusion Proteins; C021570:methylaspartate mutase; C000731191:component S, glutamate mutase protein, Bacteria; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)