Regulation of the glutamate racemase of Escherichia coli investigated by site-directed mutagenesis.
The biosynthesis of D-glutamic acid, one of the essential components of bacterial cell-wall peptidoglycan, is catalyzed by a glutamate racemase in Escherichia coli. While the other reported glutamate racemases from various (essentially gram-positive) bacterial species did not require any specific activator, the E. coli enzyme absolutely requires the presence of the peptidoglycan precursor UDP-N-acetylmuramyl-L-alanine to catalyze the interconversion of glutamic acid isomers. A comparison of the amino acid sequences of these different enzymes was made to identify amino acid residues from the E. coli enzyme that are involved in the catalysis or binding to the activator. Site-directed mutagenesis experiments are described that demonstrate the participation of cysteines 96 and 208 in the two-base reaction mechanism of the enzyme. The construction of N- or C-terminal-truncated enzymes is also described. The attractive hypothesis that the characteristic N-terminal amino acid extension (20 residues) of the E. coli enzyme could be involved in its activation by the nucleotide precursor is disproved by these experiments.
Doublet
P
P
Laboratoire des Enveloppes Bactériennes et Peptides, URA 1131 CNRS, Université Paris-sud, Orsay, France.
van Heijenoort
J
J
Mengin-Lecreulx
D
D
D000591:Amino Acid Isomerases / Q000096:biosynthesis* / Q000235:genetics*; D000595:Amino Acid Sequence; D003470:Culture Media; D003545:Cysteine / Q000378:metabolism; D004269:DNA, Bacterial / Q000032:analysis / Q000096:biosynthesis; D004789:Enzyme Activation / Q000502:physiology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D007778:Lactobacillus / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010376:Pediococcus / Q000201:enzymology / Q000235:genetics; D010957:Plasmids / Q000235:genetics
D003470:Culture Media; D004269:DNA, Bacterial; D000591:Amino Acid Isomerases; C062292:glutamate racemase; D003545:Cysteine