5.3.2.6 A33D the mutant shows highest enantioselectivity for the Michael-type addition of acetaldehyde to trans-beta nitrostyrene producing 4-nitro-3-phenylbutanal, compared to the wild type enzyme 748742 5.3.2.6 A33E the mutation significantly (4fold) improves the activity of the enzyme for the Michael-type addition of acetaldehyde to trans-beta nitrostyrene, compared to the wild type enzyme 748742 5.3.2.6 alphaI52E site-directed mutagenesis, active site mutant,the mutant shows improved trans-3-chloroacrylic acid dehalogenase activity with a 36fold increase in kcat/Km, largely due to a 110fold decrease in Km, and diminished 4-oxalocrotonate tautomerase activity. The negatively charged group may hinder the formation of the enolate intermediate and may contribute to a decrease in kcat -, 718844 5.3.2.6 alphaL8R site-directed mutagenesis, active site mutant, the mutant shows improved trans-3-chloroacrylic acid dehalogenase activity with a 50fold increase in kcat/Km, primarily from an 8.8fold increase in kcat, and diminished 4-oxalocrotonate tautomerase activity with a 5fold decrease in kcat/Km. The increased CaaD activity of L8R-4-OT does not substantially diminish the original 4-OT activity -, 718844 5.3.2.6 alphaL8R/I52E site-directed mutagenesis, active site mutant, the mutant shows improved trans-3-chloroacrylic acid dehalogenase activity with a 32fold increase in kcat/Km, largely due to a 23fold decrease in Km, and diminished 4-oxalocrotonate tautomerase activity with a 1700fold decrease in kcat/Km -, 718844 5.3.2.6 alphaR12A site-directed mutagenesis, the mutant shows highly reduced catalytic efficiency compared to the wild-type hh4-OT -, 718874 5.3.2.6 alphaR40A site-directed mutagenesis, the mutant shows reduced catalytic efficiency compared to the wild-type hh4-OT -, 718874 5.3.2.6 betaP1A site-directed mutagenesis, the mutant shows highly reduced catalytic efficiency compared to the wild-type hh4-OT -, 718874 5.3.2.6 betaR11A site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type hh4-OT -, 718874 5.3.2.6 betaR39A site-directed mutagenesis, the mutant shows reduced catalytic efficiency compared to the wild-type hh4-OT -, 718874 5.3.2.6 H6M the mutant has about 3fold increased specific activity compared with that of wild type enzyme 748742 5.3.2.6 H6M/A33E/F50V the mutation strongly enhances the activity for the Michael-type addition of butanal to trans-beta nitrostyrene, compared to the wild type enzyme 748742 5.3.2.6 M45Y/F50A the mutant enzyme displays low Michael-type addition activity compared to the wild type enzyme 748742 5.3.2.6 P1A inactive -, 749336 5.3.2.6 P1A site-directed mutagenesis, inactive mutant 718847 5.3.2.6 P1A the mutation results in 430fold decreases in kcat/Km compared to the wild type enzyme 726964 5.3.2.6 R11A site-directed mutagenesis, inactive mutant 718847 5.3.2.6 R11A site-directed mutagenesis, no kinetic effects of the R11A mutation, the stereoselectivity of the R11A-catalyzed protonation at C-5 of the dicarboxylate substrate decreases, while the stereoselectivity of protonation at C-3 of the monocarboxylate substrate increases in comparison with wild-type 4-OT -, 718830 5.3.2.6 R11A the mutation results in 280fold decreases in kcat/Km compared to the wild type enzyme 726964 5.3.2.6 R11A/R39A site-directed mutagenesis, inactive mutant -, 718830 5.3.2.6 R35A the mutation disrupts catalysis and impairs protein multimerisation -, 749336 5.3.2.6 R35Q the mutation disrupts catalysis and impairs protein multimerisation -, 749336 5.3.2.6 R39A site-directed mutagenesis, with 2-hydroxymuconate the R39A mutant shows decreased kcat by 125fold and increased Km by 1.5fold -, 718830 5.3.2.6 R39A the mutation results in 8000fold decreases in kcat/Km compared to the wild type enzyme 726964 5.3.2.6 R39E the mutant shows highest enantioselectivity for the Michael-type addition of butanal to trans-beta nitrostyrene, producing 2-ethyl-4-nitro-3-phenylbutanal, compared to the wild type enzyme 748742 5.3.2.6 R39Q site-directed mutagenesis, with 2-hydroxymuconate the R39Q mutant shows decreased kcat by 389fold and increased Km by 2.6fold, only the tight binding sites function catalytically in the R39Q mutant, structural changes in the R39Q mutant were mainly in the beta-hairpin from residues 50 to 57 which covers the active site -, 718830 5.3.2.6 R39Q titration with cis,cis-muconate shows negative cooperativity 718840 5.3.2.6 R61A site-directed mutagenesis, Arg61 mutation does not affect either substrate binding or catalysis -, 718830 5.3.2.6 R61A the mutation results in 8fold decreases in kcat/Km compared to the wild type enzyme 726964