Bioscience Biotechnology and Biochemistry · 2003 · 37 citations · 14 references
EngineeringBioengineeringBiotechnologyDirected EvolutionEnzymologyProtein EngineeringDirected Evolution Of EnzymesEnzyme EvolutionMetabolic Pathway EngineeringMutagenesis StrategiesProtein DesignProtein Engineering Of Enzymes5-Enolpyruvylshikimate-3-phosphate Synthase Mutation5-Enolpyruvylshikimate-3-phosphate Synthase ToleranceAmino Acid SubstitutionAmino Acid Substitution EffectsBacterial Resistance To GlyphosateEscherichia ColiGlyphosate Resistance MechanismsRational Design
Mutants of class I enolpyruvylshikimate 3-phosphate synthase (EPSPS) with resistance to glyphosate were produced in a previous study using the staggered extension process with aroA genes from S. typhimurium and E. coli. Two of these mutants shared a common amino acid substitution, T42M, near the hinge region between the large globular domains of EPSPS. Using site-directed mutagenisis, we produced the T42M mutants without the other amino acid changes of the original mutants. The T42M substitution alone produced enzymes with a 9- to 25-fold decreased K(m)[PEP] and a 21- to 26-fold increased K(i)[glyphosate] compared to the wild-type enzymes. These results provide more testimony for the powerful approach for protein engineering by the combination of directed evolution and rational design.
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An improved assay for nanomole amounts of inorganic phosphate
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Molecular evolution by staggered extension process (StEP) in vitro recombination
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