Purification, cloning, and cofactor independence of glutamate racemase from Lactobacillus.
Glutamate racemase has been purified more than 12,000-fold from Lactobacillus fermenti. The racemase gene has been cloned using standard hybridization techniques combined with a novel selection for in vivo glutamate racemase activity, and the racemase has been expressed in Escherichia coli as 20-25% of the total soluble cell protein. The cloned gene product is indistinguishable from that purified from Lactobacillus and is a monomer of M(r) 28,300. Both a coupled enzymatic assay and a circular dichroism assay show that the enzyme follows Michaelis-Menten kinetics, with a Km of 0.3 mM and a kcat of 70 s-1 in each reaction direction. Investigations into the cofactor dependence of glutamate racemase indicate that the enzyme employs neither pyridoxal phosphate nor a pyruvoyl group in the labilization of the proton at the stereogenic center of glutamate. Furthermore, the racemase activity is unaffected by the presence of the metal-chelating reagent EDTA. The gene sequence of the racemase is 24% identical to that of aspartate racemase from Streptococcus thermophilus and 30% identical to that of an unidentified open reading frame in the rrnB ribosomal RNA operon of E. coli. Because the two cysteine residues in glutamate racemase and their surrounding regions are well-conserved in both of these sequences, and since glutamate racemase is stabilized by the presence of reduced thiols, these residues are possible candidates for the enzymic bases that deprotonate glutamate at C-2.
Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138.
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Knowles
D000591:Amino Acid Isomerases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular / Q000379:methods; D017886:Durapatite; D005798:Genes, Bacterial; D006882:Hydroxyapatites; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016366:Open Reading Frames; D009876:Operon; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D013291:Streptococcus / Q000201:enzymology / Q000235:genetics
D006882:Hydroxyapatites; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D017886:Durapatite; D000591:Amino Acid Isomerases; C062292:glutamate racemase