Site-directed mutagenesis of putative active site residues of 5-enolpyruvylshikimate-3-phosphate synthase.
The site-directed mutagenesis of a number of proposed active site residues of 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase is reported. Several of these mutations resulted in complete loss of enzyme activity indicating that these residues are probably involved with catalysis, notably K22R, K411R, D384A, R27A, R100A, and D242A. Of those, K22R, R27A, and D384A did not bind either the substrate shikimate-3-phosphate (S3P) or glyphosate (GLP). The K411R and D242A mutants bind S3P only in the presence of GLP. The kinetic characterization of mutants R100K, K340R, and E418A, which retain activity, is reported. Of those, R100K and K340R do not accumulate enzyme intermediate of enzyme-bound product under equilibrium conditions. These residues, while not essential for catalysis, are most likely important for substrate binding. All of the mutants are shown to be correctly folded by NMR spectroscopy.
Shuttleworth
W A
WA
Department of Biochemistry and Biophysics, Washington State University, Pullman 99164-4660, USA.
Pohl
M E
ME
Helms
G L
GL
Jakeman
D L
DL
Evans
J N
JN
D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D000409:Alanine / Q000235:genetics; D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000235:genetics*; D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D008239:Lysine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry
D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D001120:Arginine; D019883:Alkyl and Aryl Transferases; D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D008239:Lysine; D000409:Alanine