5.1.2.2 A25V Catalytic efficiencies (kcat/Km) for all mutants are reduced between 6- and 40fold with the exception of V22I, V26A, V29L, and V22I/V29L which have near wildtype efficiencies with mandelate 690962 5.1.2.2 A25V site-directed mutagenesis 726974 5.1.2.2 A25V variation of the hydrophobic loop, decrease in catalytic efficiency 690962 5.1.2.2 C92S/C264S/K166C site-directed mutagenesis 727042 5.1.2.2 D270N structure of D270N with (S)-atrolactate bound in the active site reveals no geometric alterations when compared to the structure of the wild type enzyme complexed with (S)-atrolactate, with the exception that the side chain of His297 is tilted and displaced about 0.5A away from Asn270 and towards the (S)-atrolactate. The turnover number for both (R)-mandelate and (S)-mandelate are reduced 10000fold 285178 5.1.2.2 E317Q E317Q with 3400fold reduced turnover number for (R)-mandelate and 29000fold reduced turnover number for (S)-mandelate. E317Q mutant enzyme does not catalyze detectable elimination of Br- from either enantiomer of p-(bromomethyl)mandelate. E317Q mutant enzyme is irreversibly inactivated by racemic alpha-phenylglycidate at a rate comparable to that measured for wild type enzyme 285194 5.1.2.2 F52W compared to wild-type enzyme the catalytic preference of the mutant enzyme is reversed and catalytic efficiency is reduced. Mutant enzyme exhibits higher affinity for (R)-mandelate than for (S)-mandelate, and a higher turnover number with (S)-mandelate as the substrate, relative to that with (R)-mandelate 661212 5.1.2.2 F52W/Y54W compared to wild-type enzyme the catalytic preference of the mutant enzyme is reversed and catalytic efficiency is reduced. Mutant enzyme exhibits higher affinity for (R)-mandelate than for (S)-mandelate, and a higher turnover number with (S)-mandelate as the substrate, relative to that with (R)-mandelate 661212 5.1.2.2 H297N H297N has no detectable mandelate racemase activity. However, H297N catalyzes the stereospecific elimination of Br- from racemic p-(bromomethyl)mandelate to give p-(methyl)benzoylformate in 45% yield at a rate equal to that measured for wild type enzyme 285186 5.1.2.2 H297N H297N, which is inactive as a racemase catalyzes the stereospecific exchange of the alpha-proton of S- but not R-mandelate with solvent D2O at a rate that is 30% of that of the wild type enzyme 285189 5.1.2.2 H297N site-directed mutagenesis, analysis of ligand binding, kinetics 747095 5.1.2.2 K166C site-directed mutagenesis, analysis of ligand binding, kinetics 747095 5.1.2.2 K166E K166R retains low level of racemase activity. K166R mutant catalyzes the elimination of Br- from only the (R)-enantiomer of (R,S)-p-(bromomethyl)mandelate 285192 5.1.2.2 K166M site-directed mutagenesis, analysis of ligand binding, kinetics 747095 5.1.2.2 K166M/H297N site-directed mutagenesis, analysis of ligand binding, kinetics 747095 5.1.2.2 additional information Comparison of the binding affinities of the mutant variants with the intermediate/transition state analogues benzohydroxamate and cyclohexanecarbohydroxamate reveals that cationPi/N-Pi interactions between His297 and the hydroxamate/hydroximate moiety and the phenyl ring of benzohydroxamate contribute approximately 0.26 and 0.91 kcal/mol to binding, respectively, while interactions with Lys166 contribute approximately 1.74 and 1.74 kcal/mol, respectively. Lys166 contributes over 2.93 kcal/mol to the binding of (R)-atrolactate, and His297 contributes 2.46 kcal/mol to the binding of (S)-atrolactate 747095 5.1.2.2 additional information development and evaluation of a virtual mutant screening method for non-natural substrates based on the binding energy in the transition state, the method is beneficial in enzyme rational redesign and helps to better understand the catalytic properties of the enzyme, and it is effective in predicting the trend of mutational effects on catalysis, molecular dynamic simulation, overview 727384 5.1.2.2 additional information directed evolution of mandelate racemase by a high-throughput screening method, overview -, 746856 5.1.2.2 additional information evaluation of design of mandelate racemase with higher stability, usage of structural enzyme analysis for reengineering of mandelate racemase for enhanced thermal stability 742923 5.1.2.2 additional information mandelate racemase and mandelate dehydrogenase are coexpressed in Escherichia coli for the synthesis of benzoylformate by converting racemic mandelate -, 727197 5.1.2.2 N197A Kcat for (R)-mandelate reduced 30fold, Kcat for (S)-mandelate reduced 179fold relative to wild-type 285167 5.1.2.2 N197A slight activating effect of sucrose on mutant enzyme efficiency. In presewnce of polymeric viscosogens poly(ethylene glycol) and Ficoll, no effect on turnover number or the ratio of turnover number and Km-value for the wild-type enzyme is observed 650147 5.1.2.2 N197A turnover number is reduced 30fold for (R)-mandelate and 179fold for (S)-mandelate relative to wild-type enzyme. The ratio of turnover number to Km-value is reduced 208fold for (R)-mandelate and 556fold for (S)-mandelate, 3.5fold reduction in affinity for the substrate analogue (R)-atrolactate, but 51fold and 18fold reduction in affinity for alpha-hydroxybenzylphosphonate and benzohydroxamate, respectively 285195 5.1.2.2 S139A site-directed mutagenesis, the mutation leads to a significant reduction of catalytic efficiency by about 45fold and 60fold in R to S and S to R directions 728000 5.1.2.2 T24S Catalytic efficiencies (kcat/Km) for all mutants are reduced between 6- and 40fold with the exception of V22I, V26A, V29L, and V22I/V29L which have near wildtype efficiencies with mandelate 690962 5.1.2.2 T24S site-directed mutagenesis 726974 5.1.2.2 T24S variation of the hydrophobic loop, decrease in catalytic efficiency 690962 5.1.2.2 V22A Catalytic efficiencies (kcat/Km) for all mutants are reduced between 6- and 40fold with the exception of V22I, V26A, V29L, and V22I/V29L which have near wildtype efficiencies with mandelate 690962 5.1.2.2 V22A site-directed mutagenesis 726974 5.1.2.2 V22A variation of the hydrophobic loop, decrease in catalytic efficiency 690962 5.1.2.2 V22F Catalytic efficiencies (kcat/Km) for all mutants are reduced between 6- and 40fold with the exception of V22I, V26A, V29L, and V22I/V29L which have near wildtype efficiencies with mandelate 690962 5.1.2.2 V22F site-directed mutagenesis 726974 5.1.2.2 V22F variation of the hydrophobic loop, decrease in catalytic efficiency 690962 5.1.2.2 V22I Catalytic efficiencies (kcat/Km) for all mutants are reduced between 6- and 40fold with the exception of V22I, V26A, V29L, and V22I/V29L which have near wildtype efficiencies with mandelate 690962 5.1.2.2 V22I site-directed mutagenesis 726974 5.1.2.2 V22I site-directed mutagenesis, kinetic analysis -, 746856 5.1.2.2 V22I variation of the hydrophobic loop. For mandelate, catalytic efficiency similar to wild-type. For substrate 2-naphthylglycolate, increase in catalytic efficiency 690962 5.1.2.2 V22I/V29I site-directed mutagenesis, kinetic analysis 746856 5.1.2.2 V22I/V29I/Y54F site-directed mutagenesis, the mutant shows 3.5fold greater relative activity as compared to the wild-type enzyme. The enhanced catalytic efficiency mainly arises from the elevated kcat, kinetic analysis -, 746856 5.1.2.2 V22I/V29L Catalytic efficiencies (kcat/Km) for all mutants are reduced between 6- and 40fold with the exception of V22I, V26A, V29L, and V22I/V29L which have near wildtype efficiencies with mandelate 690962 5.1.2.2 V22I/V29L variation of the hydrophobic loop, catalytic efficiency similar to wild-type 690962 5.1.2.2 V22I/Y54F site-directed mutagenesis, kinetic analysis 746856 5.1.2.2 V26A Catalytic efficiencies (kcat/Km) for all mutants are reduced between 6- and 40fold with the exception of V22I, V26A, V29L, and V22I/V29L which have near wildtype efficiencies with mandelate 690962 5.1.2.2 V26A site-directed mutagenesis 726974 5.1.2.2 V26A variation of the hydrophobic loop, catalytic efficiency similar to wild-type 690962 5.1.2.2 V26A/V29L Catalytic efficiencies (kcat/Km) for all mutants are reduced between 6- and 40fold with the exception of V22I, V26A, V29L, and V22I/V29L which have near wildtype efficiencies with mandelate 690962 5.1.2.2 V26A/V29L site-directed mutagenesis 726974 5.1.2.2 V26A/V29L variation of the hydrophobic loop, decrease in catalytic efficiency 690962 5.1.2.2 V26F Catalytic efficiencies (kcat/Km) for all mutants are reduced between 6- and 40fold with the exception of V22I, V26A, V29L, and V22I/V29L which have near wildtype efficiencies with mandelate 690962 5.1.2.2 V26F site-directed mutagenesis 726974 5.1.2.2 V26F variation of the hydrophobic loop, decrease in catalytic efficiency 690962 5.1.2.2 V26I site-directed mutagenesis, the mutant shows 2fold higher catalytic efficiency towards R-mandelamide than the wild-type enzyme 727384 5.1.2.2 V26I/Y54V site-directed mutagenesis, the mutant shows 5.2fold higher catalytic efficiency towards (3R)-3-chloromandelic acid than the wild-type enzyme 727384 5.1.2.2 V26L Catalytic efficiencies (kcat/Km) for all mutants are reduced between 6- and 40fold with the exception of V22I, V26A, V29L, and V22I/V29L which have near wildtype efficiencies with mandelate 690962 5.1.2.2 V26L site-directed mutagenesis 726974 5.1.2.2 V26L variation of the hydrophobic loop, decrease in catalytic efficiency 690962 5.1.2.2 V29A Catalytic efficiencies (kcat/Km) for all mutants are reduced between 6- and 40fold with the exception of V22I, V26A, V29L, and V22I/V29L which have near wildtype efficiencies with mandelate 690962 5.1.2.2 V29A site-directed mutagenesis 726974 5.1.2.2 V29A variation of the hydrophobic loop, decrease in catalytic efficiency 690962 5.1.2.2 V29F Catalytic efficiencies (kcat/Km) for all mutants are reduced between 6- and 40fold with the exception of V22I, V26A, V29L, and V22I/V29L which have near wildtype efficiencies with mandelate 690962 5.1.2.2 V29F site-directed mutagenesis 726974 5.1.2.2 V29F variation of the hydrophobic loop, decrease in catalytic efficiency 690962 5.1.2.2 V29I site-directed mutagenesis, kinetic analysis -, 746856 5.1.2.2 V29I/Y54F site-directed mutagenesis, kinetic analysis 746856 5.1.2.2 V29L Catalytic efficiencies (kcat/Km) for all mutants are reduced between 6- and 40fold with the exception of V22I, V26A, V29L, and V22I/V29L which have near wildtype efficiencies with mandelate 690962 5.1.2.2 V29L site-directed mutagenesis 726974 5.1.2.2 V29L site-directed mutagenesis, the mutant shows 2fold higher catalytic efficiency towards R-2-naphthylglycolate than the wild-type enzyme 727384 5.1.2.2 V29L variation of the hydrophobic loop. For mandelate, catalytic efficiency similar to wild-type. For substrate 2-naphthylglycolate, increase in catalytic efficiency 690962 5.1.2.2 Y54F site-directed mutagenesis 726974 5.1.2.2 Y54F site-directed mutagenesis, kinetic analysis -, 746856 5.1.2.2 Y54L site-directed mutagenesis 726974 5.1.2.2 Y54Q compared to wild-type enzyme the catalytic preference of the mutant enzyme is reversed and catalytic efficiency is reduced. Mutant enzyme exhibits higher affinity for (R)-mandelate than for (S)-mandelate, and a higher turnover number with (S)-mandelate as the substrate, relative to that with (R)-mandelate 661212