Site-directed mutagenesis of a conserved region of the 5-enolpyruvylshikimate-3-phosphate synthase active site.
The active site of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) has been probed using site-directed mutagenesis and inhibitor binding techniques. Replacement of a specific glycyl with an alanyl or a prolyl with a seryl residue in a highly conserved region confers glyphosate tolerance to several bacterial and plant EPSPS enzymes, suggesting a high degree of structural conservation between these enzymes. The glycine to alanine substitution corresponding to Escherichia coli EPSPS G96A increases the Ki(app) (glyphosate) of petunia EPSPS 5000-fold while increasing the Km(app)(phosphoenolpyruvate) about 40-fold. Substitution of this glycine with serine, however, abolishes EPSPS activity but results in the elicitation of a novel EPSP hydrolase activity whereby EPSP is converted to shikimate 3-phosphate and pyruvate. This highly conserved region is critical for the interaction of the phosphate moiety of phosphoenolpyruvate with EPSPS.
Padgette
S R
SR
Monsanto Agricultural Company, New Products Division, Chesterfield, Missouri.
Re
D B
DB
Gasser
C S
CS
Eichholtz
D A
DA
Frazier
R B
RB
Hironaka
C M
CM
Levine
E B
EB
Shah
D M
DM
Fraley
R T
RT
Kishore
G M
GM
D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology / Q000235:genetics; D001665:Binding Sites; D005075:Biological Evolution; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010944:Plants / Q000201:enzymology / Q000235:genetics; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D014166:Transferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D014166:Transferases; D019883:Alkyl and Aryl Transferases; D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase