Do closer mutations improve enzyme properties better than distant mutations?

Krista L. Morley and Romas J. Kazlauskas

Supplementary Material

Table 1. Summary of Random Mutagenesis Literature Data for Enantioselectivity (Figure 1a).a

Enzyme / Mutation / Distance, Å / E / G‡(kcal/mol) / Ref.
Pseudomonas fluorescens esterase / T230I / 14 / 12 to 19 ((methyl 3-bromo-2-methylpropanoate) / 0.27 / 1
Pseudomonas fluorescens esterase / T230P / 14 / 12 to 17 ((methyl 3-bromo-2-methylpropanoate) / 0.21 / 1
Pseudomonas fluorescens esterase / D158N / 13 / 3.5 to 5.8 (ethyl 3-phenylbutyrate) / 0.30 / 1
Pseudomonas fluorescens esterase / D158E / 13 / 3.5 to 8 (ethyl 3-phenylbutyrate) / 0.49 / 1
Pseudomonas fluorescens esterase / D158L / 13 / 3.5 to 12 (ethyl 3-phenylbutyrate) / 0.73 / 1
Pseudomonas fluorescens esterase / D158C / 13 / 3.5 to 8 (ethyl 3-phenylbutyrate) / 0.49 / 1
Pseudomonas fluorescens esterase / D158F / 13 / 3.5 to 9 (ethyl 3-phenylbutyrate) / 0.56 / 1
Pseudomonas fluorescens esterase / L181Q / 12 / 3.5 to 6.6 (ethyl 3-phenylbutyrate) / 0.38 / 1
Pseudomonas fluorescens esterase / L181W / 12 / 3.5 to 8 (ethyl 3-phenylbutyrate) / 0.49 / 1
Pseudomonas fluorescens esterase / L181T / 12 / 3.5 to 9 (ethyl 3-phenylbutyrate) / 0.56 / 1
Pseudomonas fluorescens esterase / L181S / 12 / 3.5 to 10 (ethyl 3-phenylbutyrate) / 0.62 / 1
Pseudomonas putida benzoylformate decarboxylase / L476K / 5 / 12 to 66 (2-hydroxy-1-phenylpropanone) / 0.99 / 2
Pseudomonas putida benzoylformate decarboxylase / L476P,S, M,G / 5 / 12 to 32 (2-hydroxy-1-phenylpropanone) / 0.57 / 2
Pseudomonas putida benzoylformate decarboxylase / L476Q,C / 5 / 12 to 39 (2-hydroxy-1-phenylpropanone) / 0.68 / 2
Pseudomonas putida benzoylformate decarboxylase / L476H / 5 / 12 to 28 (2-hydroxy-1-phenylpropanone) / 0.49 / 2
Pseudomonas putida benzoylformate decarboxylase / L476A / 5 / 12 to 49 (2-hydroxy-1-phenylpropanone) / 0.78 / 2
Arthrobacter sp. DSM 9771 hydantoinase / V154A / 10.6b / 2.7 to 28 (5-(2-methylthioethyl)hydantoin) / 1.36 / 3
Arthrobacter sp. DSM 9771 hydantoinase / I95F / 9.8b / 0.4 to 1.6 (L-5-(2-methylthioethyl)hydantoin) / 0.86 / 3
Arthrobacter sp. DSM 9771 hydantoinase / I95L / 9.8b / 0.4 to 0.6 (L-5-(2-methylthioethyl)hydantoin) / 0.31 / 3
Pseudomonas aeruginosa lipase / S155F / 11.3 / 1.1 to 9.5 (p-nitrophenyl 2-methyldecanoate) / 1.28 / 4
Pseudomonas aeruginosa lipase / S155F / 11.3 / 4.4 to 5.7 (p-nitrophenyl 2-methyldecanoate); in generation 2 / 0.15 / 4
Pseudomonas aeruginosa lipase / S155F / 11.3 / 9.7 to 20.5 (p-nitrophenyl 2-methyldecanoate); in generation 3 / 0.44 / 4
Pseudomonas aeruginosa lipase / S155F / 11.3 / 10.7 to 14.7 (p-nitrophenyl 2-methyldecanoate); in generation 4 / 0.19 / 4
Pseudomonas aeruginosa lipase / S155F / 11.3 / 12 to 20.1 (p-nitrophenyl 2-methyldecanoate); in generation 4 / 0.31 / 4
Pseudomonas aeruginosa lipase / S155L / 11.3 / 1.1 to 4.4 (p-nitrophenyl 2-methyldecanoate) / 0.44 / 4
Pseudomonas aeruginosa lipase / F259L / 19.3 / 9.7 to 10.7 (p-nitrophenyl 2-methyldecanoate) / 0.06 / 4
Pseudomonas aeruginosa lipase / L110R / 12.9c / 10.7 to 12 (p-nitrophenyl 2-methyldecanoate) / 0.07 / 4
Pseudomonas aeruginosa lipase / S149G / 22.1 / 1.1 to 2.1 (p-nitrophenyl 2-methyldecanoate) / 0.38 / 4
Pseudomonas aeruginosa lipase / V47G / 13.7 / 4.4 to 9.7 (p-nitrophenyl 2-methyldecanoate) / 0.47 / 4
Pseudomonas aeruginosa lipase / V47G / 13.7 / 5.7 to 20.5 (p-nitrophenyl 2-methyldecanoate) / 0.76 / 4
Pseudomonas aeruginosa lipase / L162G / 6.4 / 1.1 to 34 (p-nitrophenyl 2-methyldecanoate) / 2.03 / 5

a Criteria: Only increases in enantioselectivity caused by single mutations where the structure of the enzyme is known. Free energy values are calculated at 298 K. Distances are from the stereocenter of the substrate to the alpha carbon of the mutated amino acid.

b Distance to active site Zn1.

cResidue 110 in protein data bank file 1ex9 is Lys not Leu; however this is the distance to Leu 118.

Table 2. Summary of Rational Design Literature Data for Enantioselectivity (Figure 1a).a

Enzyme / Mutation / Distance, Å / E / G‡(kcal/mol) / Ref.
Agrobacterium radiobacter AD1 epoxide hydrolase / Y215F / 9.8b / 100 to 200 (p-nitrostyrene oxide) / 0.41 / 6
Agrobacterium radiobacter AD1 epoxide hydrolase / Y215F / 9.8b / 16 to 30 (styrene oxide) / 0.37 / 6
Agrobacterium radiobacter AD1 epoxide hydrolase / Y215F / 9.8b / 6.5 to 12 (m-chlorostyrene oxide) / 0.36 / 6
Agrobacterium radiobacter AD1 epoxide hydrolase / Y215F / 9.8b / 32 to 130 (p-chlorostyrene oxide) / 0.83 / 6
Agrobacterium radiobacter AD1 epoxide hydrolase / Y215H / 9.8b / 3.4 to 13 (p-nitrophenyl glycidyl ether) / 0.79 / 7
Agrobacterium radiobacter AD1 epoxide hydrolase / Y215H / 9.8b / 56 to 156 (p-nitrostyrene oxide) / 0.61 / 7
Agrobacterium radiobacter AD1 epoxide hydrolase / F108I / 3.8b / 3.4 to 20 (p-nitrophenyl glycidyl ether) / 1.05 / 7
Candida antarctica lipase B / S47A / 9.2 / 6.5 to 12.5 (1-bromo-2-octanol) / 0.39 / 8
Candida antarctica lipase B / S47A / 9.2 / 14 to 28 (1-chloro-2-octanol) / 0.41 / 8
Candida antarctica lipase B / T40A / 5.5 / 1.6 to 9.8 (ethyl 2-hydroxypropanoate) / 1.07 / 9
Candida antarctica lipase B / T40V / 5.5 / 1.6 to 22 (ethyl 2-hydroxypropanoate) / 1.55 / 9
Themomyces lanuginosa lipase / E87A / 13.5c / 10.5 to 17.4 (heptyl 2-methyldecanoate) / 0.30 / 10
Pseudomonas diminuta phosphotriesterase / G60A / 7.6 / 1 to 11 (methyl ethyl p-nitrophenylphosphate) / 1.42 / 11
Pseudomonas diminuta phosphotriesterase / G60A / 7.6 / 32 to 400 (methyl isopropyl p-nitrophenylphosphate) / 1.50 / 11
Pseudomonas diminuta phosphotriesterase / G60A / 7.6 / 93 to 13,000 (methyl phenyl p-nitrophenylphosphate) / 2.93 / 11
Pseudomonas diminuta phosphotriesterase / G60A / 7.6 / 10 to 24 (ethyl isopropyl p-nitrophenylphosphate) / 0.52 / 11
Pseudomonas diminuta phosphotriesterase / G60A / 7.6 / 21 to 11,000 (ethyl phenyl p-nitrophenylphosphate) / 3.71 / 11
Pseudomonas diminuta phosphotriesterase / G60A / 7.6 / 35 to 15,000 (isopropyl phenyl p-nitrophenylphosphate) / 3.59 / 11
Rhizopus oryzae lipase / L258F / 6.4 / 5 to 14 (1,3-dioleoyl-2-deoxy-2-palmitoylaminolglycerol) / 0.61 / 12
Rhizopus oryzae lipase / L258F / 6.4 / 8 to 22 (1,3-dioleoyl-2-deoxy-2-phenylglycerol) / 0.60 / 12
horseradish peroxidase / F41L / 7.7d / 2.1 to 32 (phenyl ethylsulfide) / 1.61 / 13
horseradish peroxidase / F41L / 7.7d / 1.2 to 32 (phenyl cyclopropylsulfide) / 1.94 / 13
horseradish peroxidase / F41L / 7.7d / 5.5 to 199 (2-naphthalenyl methylsulfide) / 2.12 / 13
Pseudomonas fluorescens esterase / V121S / 6.8 / 12 to 61 ((methyl 3-bromo-2-methylpropanoate) / 0.96 / 14
Pseudomonas fluorescens esterase / V121M / 6.8 / 12 to 36 ((methyl 3-bromo-2-methylpropanoate) / 0.65 / 14
Pseudomonas fluorescens esterase / W28L / 5.4 / 12 to 58 ((methyl 3-bromo-2-methylpropanoate) / 0.93 / 14
Pseudomonas fluorescens esterase / W28F / 5.4 / 12 to 32 ((methyl 3-bromo-2-methylpropanoate) / 0.58 / 14
Pseudomonas fluorescens esterase / W28Y / 5.4 / 12 to 29 ((methyl 3-bromo-2-methylpropanoate) / 0.52 / 14
Bacillus subtilis p-nitrobenzyl esterase / G105A / 5.0e / 3 to 19 (2-phenyl-3-butin-2-yl acetate) / 1.09 / 15

a Criteria: Only changes (increase or decrease) in enantioselectivity caused by single mutations where the structure of the enzyme is known. Free energy values are calculated at 298 K.

bNo model containing substrate was available; distance to closest O of active site Asp107

c No model containing substrate was available; distance to O of active site Ser146.

d Distance is to the hydroxamate oxygen of a bound inhibitor in protein data bank file 1gx2.

eNo model containing substrate was available; distance to O of active site Ser189.

References

1. Horsman, G. P. et al. (2003) Mutations in distant residues moderately increase the enantioselectivity of Pseudomonas fluorescens esterase toward methyl 3-bromo-2-methylpropanonate and ethyl 3-phenylbutyrate. Chem. Eur. J. 9, 1933-1939

2. Lingen, B. et al. (2002) Improving the carboligase activity of benzoylformate decarboxylase from Pseudomonas putida by a combination of directed evolution and site-directed mutagenesis. Protein Eng. 15, 585-593

3. May, O. et al. (2000) Inverting enantioselectivity by directed evolution of hydantoinase for improved production of l-methionine. Nature Biotechnol. 18, 317-320

4. Liebeton, K. et al. (2000) Directed evolution of an enantioselective lipase. Chem. Biol. 7, 709-715

5.Reetz, M. T. et al. (2001) Directed evolution of an enantioselective enzyme through combinatorial multiple- cassette mutagenesis. Angew. Chem. Int. Ed., 40, 3589-3591

6. Rink, R. et al. (1999) Mutation of tyrosine residues involved in the alkylation half reaction of epoxide hydrolase from Agrobacterium radiobacter AD1 results in improved enantioselectivity. J. Am. Chem. Soc. 121, 7417-7418

7. van Loo, B. et al. (2004) Directed evolution of epoxide hydrolase from A. radiobacter toward higher enantioselectivity by error-prone PCR ad DNA shuffling. Chem. Biol. 11, 981-990

8. Rotticci, D. et al. (2001) Improved enantioselectivity of a lipase by rational protein engineering. ChemBioChem 2, 766-770

9. Magnusson, A. et al. (2001) Creation of an enantioselective hydrolase by engineered substrate-assisted catalysis. J. Am. Chem. Soc. 123, 4354-4355

10. Holmquist, M. et al. (1993) Lipases from Rhizomucor miehei and Humicola lanuginosa: modification of the lid covering the active site alters enantioselectivity. J. Prot. Chem. 12, 749-757

11. Chen-Goodspeed, M. et al. (2001) Structural determinants of the substrate and stereochemical specificity of phosphotriesterase. Biochemistry 40, 1325-1331

12. Scheib, H. et al. (1998) Rational design of Rhizopus oryzae lipase with modified stereoselectivity toward triradylglycerols. Protein Eng. 11, 675-682

13. Ozaki, S.-I. and Ortiz de Montellano, P. R. (1994) Molecular engineering of horseradish peroxidase. Highly enantioselective sulfoxidation of aryl alkyl sulfides by the Phe-41Leu mutant. J. Am. Chem. Soc. 116, 4487-4488

14. Park, S. et al. (2005) Focusing mutations into the substrate-binding site of Pseudomonas fluorescens esterase increases the enantioselectivity more effectively than random mutagenesis. Chem. Biol. 11, in press

15. Henke, E. et al. (2003) A molecular mechanism of enantiorecognition of tertiary alcohols by carboxylesterases. ChemBioChem 4, 485-493

Table 3. Summary of Mutagenesis Literature Data for Substrate Specificity (Figure 1b)a

Enzyme / Mutation / Distance, Å / S / G‡(kcal/mol) / Ref.
Pyrococcus furiosus-glucosidase (Cel B) / N415S / 12.3b / 1.9 to 12.3 (p-nitrophenyl--D-glucopyranoside/p-nitrophenyl--D-galactopyranoside) / 1.11
/ 1
Glutaryl acylase / Y178H / 12.2c / 4.8 x 10-3 to 3.6 x 10-2 (adipyl-7-ADCA/glutaryl-7-ACA) / 1.19 / 2
Glutaryl acylase / N266M / 7.1c / 2.6 x 10-3 to 0.54 (adipyl-7-ADCA/glutaryl-7-ACA) / 3.16 / 3
Glutaryl acylase / N266H / 7.1c / 2.6 x 10-3 to 4.8 x 10-2 (adipyl-7-ADCA/glutaryl-7-ACA) / 1.73 / 3
Tagatose-1,6-bisphosphate aldolase / H26Y / 11.6d / 3.0 x 10-3 to 3.5 x 10-2 (fructose-1,6-biphosphate/tagatose-1,6-biphosphate) / 1.45 / 4
Tagatose-1,6-bisphosphate aldolase / D104G / 8.2d / 3.0 x 10-3 to 6.2 x 10-2 (fructose-1,6-biphosphate/tagatose-1,6-biphosphate) / 1.79 / 4
Biphenyl dioxygenase / F227V / 9.5e / 0.05 to 11.8 (ratio of 2,3/5,6 dihydroxlation of 3,3'-dichlorobiphenyl) / 3.24 / 5
Biphenyl dioxygenase / I335F / 12.8e / 0.33 to 6.6 (ratio of 2,3/5,6 dihydroxlation of 2,2'-dichlorobiphenyl)) / 1.77 / 5
Biphenyl dioxygenase / T376N / 8.8e / 0.33 to 9.1 (ratio of 2,3/5,6 dihydroxlation of 2,2'-dichlorobiphenyl) / 1.96 / 5
Biphenyl dioxygenase / T376N / 8.8e / 0.11 to 7.9 (ratio of 3,4/5,6 dihydroxlation of 2,5,2'-trichlorobiphenyl) / 2.53 / 5
Biphenyl dioxygenase / T376N / 8.8e / 0.13 to 12.0 (ratio of 3,4/2,3 dihydroxlation of 2,5,4'-trichlorobiphenyl) / 2.68 / 5
Biphenyl dioxygenase / F377L / 8.9e / 0.33 to 8.0 (ratio of 2,3/5,6 dihydroxlation of 2, 2'-dichlorobiphenyl) / 1.89 / 5
Biphenyl dioxygenase / F377L / 8.9e / 0.11 to 18.1 (ratio of 3,4/5,6 dihydroxlation of 2,5,2'-trichlorobiphenyl) / 3.02 / 5
Biphenyl dioxygenase / F377L / 8.9e / 0.13 to 13.4 (ratio of 3,4/2,3 dihydroxlation of 2,5,4'-trichlorobiphenyl) / 2.74 / 5
Biphenyl dioxygenase / F377A / 8.9e / 0.05 to 3.8 (ratio of 2,3/5,6 dihydroxlation of 3,3'-dichlorobiphenyl) / 2.56 / 5
D-Hydantoinase / F159V / 11.3f / 0.74 to 23 (hydroxyphenylhydantoin/hydantoin) / 2.03 / 6
D-Hydantoinase / F159A / 11.3f / 0.74 to 150 (hydroxyphenylhydantoin/hydantoin) / 3.15 / 6
D-Hydantoinase / F159S / 11.3f / 0.74 to 190 (hydroxyphenylhydantoin/hydantoin) / 3.29 / 6
Alicyclobacillus acidocaldarius esterase 2 / M211S / 9.0g / 0.75 to 2.14 (pNP-decanoate/pNP-octanoate) / 0.62 / 7
Alicyclobacillus acidocaldarius esterase 2 / M211S / 9.0g / 3.47 to 11.9 (pNP-decanoate/pNP-hexanoate) / 0.73 / 7
Alicyclobacillus acidocaldarius esterase 2 / M211S / 9.0g / 17.3 to 44.6 (pNP-decanoate/pNP-butanoate) / 0.56 / 7
Castor ∆9-18:0-ACP desaturase / M114F / 14.7h / 0.07 to 4.2 (14:0-ACP/16:0-ACP) / 2.42 / 8
Subtilisin BPN' / E156S / 10.0i / 8.8 x 10-4 to 1.7 x 10-1 (succinyl-Ala-Ala-Pro-Glu-p-nitroanilide/succinyl-Ala-Ala-Pro-Lys-p-nitroanilide) / 3.12 / 9

a Criteria: Only changes in substrate specificity (S; where S is the rate of reaction of one substrate as compared with the other)caused by single mutations where the structure of the enzyme is known. Distance from C of mutated amino acid to active site residue unless otherwise specified. Free energy values are calculated at 298 K.

b Distance to closest O of active site Glu372; based on structure of -glucosidase from Bacillus Polymyxa.

cDistance to O of active site Ser199; residues are Y149, N237, S170 in protein data bank file 1fm2.

d.Distance to closest O of active site Asp82.

e. Distance to N of active site His239; based on structure of Naphthalene 1,2-Dioxygenase; Dihydroxylation ratios were calculated with dioxygenation mode transformation activities cited in the publication for wild-type and mutant enzymes. When the minor product was not detected in the reaction we assigned an activity of 1 g/20h to the enzyme based on the lower limits of activity reported by the authors. For example, a transformation activity of 18.3 g/20h is reported for the 5,6 mode of 3,3'-dichlorobiphenyl dioxygenation by the wild-type enzyme whereas an activity of 11.8 g/20h is reported for the 2,3 mode by a Phe 227Val mutant. Since the minor product is not observed in either reaction, this corresponds to a 2,3/5,6-dihdroxylation ratio of 1/18.3 (0.05) for wild-type enzyme and 11.8/1 (11.8) for Phe227Val. Consequently, the regioselectivity is altered by 235-fold (11.8/0.05) for the Phe227Val mutant.

f Distance to N of active site His60.

gDistance to O of active site Ser155.

hDistance to N of active site His232.

iDistance to Cof P1 residue of bound substrate in protein data bank file 1sua.

References

1. Lebbink, J. H. G. et al. (2000) Improving low-temperature catalysis in the hyperthermostable pyrococcus furiosus -Glucosidase CelB by directed evolution. Biochemistry 39, 3656-3665

2. Sio, C. F. et al. (2002) Directed evolution of a glutaryl acylase into an adipyl acylase. Eur. J. Biochem. 269, 4495–4504

3. Otten, L. G. et al. (2004) Mutational analysis of a key residue in the substrate specificity of a cephalosporin acylase. ChemBioChem 5, 820-825

4. Williams, G. J. et al. (2003) Modifying the stereochemistry of an enzyme-catalyzed reaction by directed evolution. Proc. Nat. Acad. Soc.USA 100, 3143-3148

5. Suenaga, H. et al. (2002) Alteration of regiospecificity in biphenyl dioxygenase by active-site engineering. J. Bacteriol. 184, 3682–3688

6. Cheon, Y.-H. et al. (2004) Manipulation of the active site loops of D-hydantoinase, a (/)8-barrel protein for modulation of the substrate specificity. Biochemistry 43, 7413-7420

7. Manco, G. et al. (2001) Residues at the active site of the esterase 2 from Alicyclobacillus

acidocaldarius involved in substrate specificity and catalytic activity at high temperature.J. Biol. Chem. 276, 37482–37490.

8. Whittle, E. and Shanklin, J. Engineering 9-16:0-acyl carrier protein (ACP) desaturase specificity based on combinatorial saturation mutagenesis and logical redesign of the castor 9-18:0-ACP desaturase. J. Biol. Chem. 276, 21500–21505

9. Wells, J. A. et al. (1987) Recruitment of substrate-specificity properties from one enzyme into a related one by protein engineering. Proc. Nat. Acad. Soc.USA 84, 5167-5171

Table 4. Summary of Mutagenesis Literature Data for Catalytic Promiscuity (Figure 1c)a

Enzyme / Mutation / Distance, Å /  kcat/KM / G‡(kcal/mol) / Ref.
L-Ala-D/L-Glu epimerase / D297G / 8.4b / 5.2 x 10-3 to 12.5 (s-1M-1; o-succinylbenzoate) / 4.61 / 1
Muconate lactonizing enzyme II / E323G / 9.0c / 1.5 x 10-3 to 1.9 x 103 (s-1M-1; o-succinylbenzoate) / 8.32 / 1
Aspartate aminotransferase / R292K / 14.7d / 1.3 x 10-4 to 20 x 10-4 [(kcat s-1) -Decarboxylation (L-aspartate)] / 1.62 / 2
Aspartate aminotransferase hexamutant / A293D / 7.4e / 370 to 1200 (s-1M-1 x 10-2;tyrosine) / 0.7 / 3
Aspartate aminotransferase hexamutant / I73V / 8.0e / 370 to 880 (s-1M-1 x 10-2;tyrosine) / 0.51 / 3
Cyclophilin / A91S / 6.8f / 10-3 to 73.1 (s-1M-1; furylacryloyl-alanylproline) / 6.63 / 4
Papain / Q19E / 9.0g / 0.03 to 1150 (s-1M-1; methoxycarbonyl-L-(phenylalanyl)-L-alanine nitrile) / 6.25 / 5
Asparagine Synthetase B / N74D / 5.2h / 0.015 to 39.3 (s-1M-1; 2-amino-4-cyanobutyric acid) / 4.66 / 6
Butylcholinesterase / G117H / 6.0i / 0.17 x 10-5 to 0.17 (s-1M-1; echothiophate) / 6.82 / 7
Butylcholinesterase / G117H / 6.0i / 0.18 x 10-5 to 0.18 (s-1M-1; paraoxon) / 6.82 / 7
N-acetylneuraminate lyase (NAL) / L142R / 11.2j / 22 to 410 (s-1M-1; L-aspartate--semialdehyde) / 1.73 / 8
Phosphotriesterase / H254R / 10.3k / 4.8 x 102 to 2.6 x 103 (s-1M-1; 2-naphthyl acetate) / 1.00 / 9
Phosphotriesterase / H254R / 10.3k / 5.2 x 102 to 3.2 x 103 (s-1M-1; carboxy fluorescein diacetate / 1.08 / 9
Candida AntarcticaLipase B / S105A / 3.8l / 0.000325 to 0.0013 (molmin-1mg-1; hexanal) / 0.82 / 10
Candida AntarcticaLipase B / S105A / 3.8l / 0.67 to 1.03 (min-1; 3-(1-methyl-butylsulfanyl)-butyraldehyde) / 0.25 / 11
Candida AntarcticaLipase B / S105A / 3.8l / 1.8 x 10-2 to 5.6 x 10-2 (min-1; 3-(1-methyl-butylsulfanyl)-pentanal) / 0.67 / 11
Candida AntarcticaLipase B / S105A / 3.8l / 9.7 x 10-2 to 0.27 (min-1; 3-(1-methyl-butylsulfanyl)-3-phenyl-propionaldehyde) / 0.61 / 11
Candida AntarcticaLipase B / S105A / 3.8l / 1.0 x 10-2 to 6.4 x 10-2 (min-1; 3-ethylsulfanyl-propionic acid methyl ester) / 1.10 / 11
Candida AntarcticaLipase B / S105A / 3.8l / 1.7 x 10-5 to 2.8 x 10-2 (min-1; 3-(1-methyl-butylsulfanyl)-propionic acid methyl ester) / 4.39 / 11
Serum paraoxonase / V346A / 7.6m / 3.5 x 103 to 1.3 x 104 (molmin-1mg-1; 7-O-diethylphosphoryl-3-cyano-7-hydroxycoumarin) / 0.78 / 12
Agrobacterium sp. -glucosidase Abg / E358A / 6.7n / glycosynthase activity / n.a. / 13
Agrobacterium sp. -glucosidase Abg / E171A / 7.1o / thioglycoligase activity / n.a. / 14
Cellulomonasfimi Man2A / E429A / 7.1p / thioglycoligase activity / n.a. / 14

a Criteria: Only changes in catalytic plasticity caused by single mutations where the structure of the enzyme is known. Distance from C of mutated amino acid to active site residue unless otherwise specified. Free energy values are calculated at 298 K. n.a. = not applicable due to lack of kinetic data.

b Distance to N of active site Lys247.

c Distance to N of active site Lys269.

d Distance to closest N of active site Arg386.

e Distance to closest N of active site Arg292.

f Distance to closest N of active site Arg48; residues are A87 and R43 in protein data bank file 1lop.

gDistance to S of active site Cys25.

h Distance to C of active site Cys1Ala.

i Distance to O of active site Ser198.

j Distance to N of active site Lys165.

k Distance to P atom of bound substrate analogue diethyl 4-methylbenzylphosphate.

l Distance to P atom of bound substrate analogue n-hexylchlorophosphonate ethyl ester.

m Distance to N of active site His115.

n Distance to closest O of active site Glu171;Based on structure of -glucosidase from Bacillus polymyxa.

o Distance to closest O of active site Glu358; Based on structure of -glucosidase from Bacillus polymyxa.

p Distance to closest O of active site Glu519; Based on structure of -glucosidase from Bacillus polymyxa.

References

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2. Vacca, R. A. et al. (1997) Active-site Arg  Lys substitutions alter reaction and substrate specificity of aspartate aminotransferase. J. Biol. Chem. 272, 21932-21937

3. Rothman, S. C. et al. (2004) Directed evolution relieves product inhibition and confers in vivo function to a rationally designed tyrosine aminotransferase. Prot. Sci. 13, 763-772

4. Quemeneur, E. et al. (1998) Engineering cyclophilin into a proline-specific endopeptidase. Nature 391, 301-304

5. Dufour, E. et al. (1995) Engineering nitrile hydratase activity into a cysteine protease by a single mutation. Biochemistry 34, 16382-16388

6. Boehlein, S. K. et al. (1997) Catalytic activity of the N-terminal domain of Escherichia coli asparagine synthetase B can be reengineered by single-point mutation. J. Am. Chem. Soc. 119, 5785-5791

7. Lockridge, O. et al. (1997) A single amino acid substitution, Gly117His, confers phosphotriesterase (organophosphorus acid anhydride hydrolase) activity on human butyrylcholinesterase. Biochemistry, 36, 786-795

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Table 5. Summary of Mutagenesis Literature Data for Catalytic Activity (Figure 2a)a

Enzyme / Mutation / Distance, Å /  kcat/KM / G‡(kcal/mol) / Ref.
Pyrococcus furiosus -glucosidase (Cel B) / N415S / 12.3b / 25 to 43 (s-1mM-1;p-nitrophenyl--D-glucopyranoside) / 0.32
/ 1
Pyrococcus furiosus -glucosidase (Cel B) / T371A / 6.4b / 0.69 to 1.4 (cellobiose) / 0.42
/ 1
Pyrococcus furiosus -glucosidase (Cel B) / E417S / 10.6b / 1.6 to 7.7 (s-1mM-1; o-nitrophenol--D-galactopyranoside-6-phosphate) / 0.93
/ 2
Haloalkane dehalogenase (DhlA) / A149T / 6.6c / 2.2 x 103 to 3.3 x 103 (s-1M-1; 1,2-dichloroethane) / 0.24 / 3
1,4--D-glucan glucohydrolase / I170T / 7.8d / 1.6 to 2.2 (s-1mM-1; cellobiose) / 0.19 / 4
E. coli galactokinase GalK / Y371H / 20.1e / 8.3 to 42.6 (mM-1min-1; 2-deoxy-D-galactose) / 0.97 / 5
E. coli galactokinase GalK / Y371H / 20.1e / 4.0 to 29.5 (mM-1min-1D-GalNH2) / 1.18 / 5
E. coli galactokinase GalK / Y371H / 20.1e / 0.8 to 6.4 (mM-1min-13-deoxy-D-galactose) / 1.23 / 5
E. coli galactokinase GalK / Y371H / 20.1e / 0.59 to 12.6 (mM-1min-16-deoxy-D-galactose) / 1.81 / 5
L. Lactis galactokinase GalK / Y385H / 20.1f / 18.1 to 35.3 (min-1mM-1; D-Gal) / 0.40 / 6
Glutaryl acylase / Y178H / 12.2g / 0.5 to 1.5 (s-1mM-1; adipyl-7-ADCA) / 0.65 / 7
Glutaryl acylase / N266M / 7.1g / 0.3 to 5.8 (s-1mM-1; adipyl-7-ADCA) / 1.75 / 8
Glutaryl acylase / N266H / 7.1g / 0.3 to 3.4 (s-1mM-1; adipyl-7-ADCA) / 1.44 / 8
-glucoronidase / T509A / 15.1h / 25 to 100 (min-1M-1) p-nitrophenyl galactoside) / 0.82 / 9
-glucoronidase / S557P / 16.9h / 25 to 50 (min-1M-1; p-nitrophenyl galactoside) / 0.41 / 9
-glucoronidase / N566S / 10.8h / 25 to 50 (min-1M-1; p-nitrophenyl galactoside) / 0.41 / 9
-glucoronidase / N566S / 10.8h / 1 to 21 (rel rates, p-nitrophenyl--D-xylopyranoside) / 1.80 / 10
Galactose oxidase / C383S / 10.3i / 16 to 55.6 (s-1M-1; D-galactose) / 0.74 / 11
Galactose oxidase / C383S / 10.3i / 17.8 to 52.4 (s-1M-1; 1-methyl--D-galactopyranoside) / 0.64 / 11
Tagatose-1,6-bisphosphate aldolase / H26Y / 11.6j / 3.2 to 89 (min-1mM-1;fructose 1,6-biphosphate) / 1.97 / 12
Tagatose-1,6-bisphosphate aldolase / D104G / 8.2j / 3.2 to 12 (min-1mM-1;fructose-1,6-biphosphate) / 0.78 / 12
Tagatose-1,6-bisphosphate aldolase / S106G / 12.1j / 3.2 to 26 (min-1mM-1;fructose-1,6-biphosphate) / 1.24 / 12
Penicillin acylase / F71C / 10.8k / 1.1x106 to 6.1x106(s-1M-1; N-(3-
Carboxy-4-nitrophenyl)phenylacetamide) / 1.01 / 13
Penicillin acylase / F71L / 10.8k / 1.1x106 to 1.1x107 (s-1M-1; N-(3-
Carboxy-4-nitrophenyl)phenylacetamide) / 1.36 / 13
Pseudomonas diminuta organophosphate hydrolase / I274T / 16.3l / 1.8x103 to 9.9x103 (s-1mM-1; Coumaphos-o-analogue) / 1.01 / 14
Pseudomonas diminuta organophosphate hydrolase / I274T / 16.3l / 9.3x102 to 3.6x103 (s-1mM-1; Methyl-paraoxon) / 0.80 / 14
Phosphotriesterase / I106S / 9.2m / 990 x105 to 1330 x105 (s-1M-1; paraoxon) / 0.17 / 15
Toluene 4-monooxygenase / I100A / 9.5n / 0.008 to 0.13 (nmol/min/mg; nitrobenzene) / 1.65 / 16
Toluene 4-monooxygenase / I100S / 9.5n / 0.008 to 0.06 (nmol/min/mg; nitrobenzene) / 1.19 / 16
Vanillyl-alcohol oxidase / I238T / 31.7o / 1 to 4.2 (s-1mM-1; creosol) / 0.85 / 17
Vanillyl-alcohol oxidase / F454Y / 16.6o / 1 to 10.8 (s-1mM-1; creosol) / 1.41 / 17
Vanillyl-alcohol oxidase / E502G / 15.6o / 1 to 3.7 (s-1mM-1; creosol) / 0.77 / 17
Vanillyl-alcohol oxidase / T505S / 12.9o / 1 to 3.9 (s-1mM-1; creosol) / 0.81 / 17
Humicola insolens Cel7B glycosynthase E197A / A197S / 9.6p / 27.8 to 964 (min-1mM-1; lactosyl fluoride) / 2.10 / 18
TEM-1 -lactamase / Y105W / 9.4q / 2.9 to 3.9x107 (s-1M-1; benzylpenicillin) / 0.18 / 19
TEM-1 -lactamase / Y105W / 9.4q / 5.5 to 8.8x105 (s-1M-1; cephalothin) / 0.28 / 19
TEMpUC19-lactamase / R164N / 13.0r / 7.4x102 to 1.2x104 (s-1M-1; Aztreonam) / 1.65 / 20
TEMpUC19-lactamase / R164N / 13.0r / 7.8x102 to 2.3x104 (s-1M-1; Cefotaxime) / 2.00 / 20
TEMpUC19-lactamase / R164N / 13.0r / 4.0x101 to 2.7x104 (s-1M-1; Ceftazidime) / 3.86 / 20
TEMpUC19-lactamase / R164N / 13.0r / 2.5 x 103 to 9.2 x 105 (s-1M-1; Cefepime) / 3.50 / 21
SHV-1 -lactamase / M69F / 5.6s / 0.014 to 0.15 (s-1M-1; Ceftazidime) / 1.40 / 22
Castor ∆9-18:0-ACP desaturase / M114F / 14.7t / 0.8 to 4.8 (nm/min/mg; 14:0-ACP) / 1.06 / 23
Castor ∆9-18:0-ACP desaturase / P179L / 17.5t / 0.8 to 15.2 (nm/min/mg; 14:0-ACP) / 1.74 / 23
Castor ∆9-18:0-ACP desaturase / P179I / 17.5t / 0.8 to 18.4 (nm/min/mg; 14:0-ACP) / 1.86 / 23
Castor ∆9-18:0-ACP desaturase / T181W / 19.0t / 0.8 to 24.8 (nm/min/mg; 14:0-ACP) / 2.03 / 23
Castor ∆9-18:0-ACP desaturase / G188L / 14.9t / 0.8 to 11.2 (nm/min/mg; 14:0-ACP) / 1.56 / 23
Castor ∆9-18:0-ACP desaturase / G188L / 14.9t / 11.5 to 172.5 (nm/min/mg; 16:0-ACP) / 1.60 / 23
D-Hydantoinase / F159V / 11.3u / 3.3 to 14 (s-1M-1; hydroxyphenylhydantoin) / 0.86 / 24
D-Hydantoinase / F159A / 11.3u / 3.3 to 15 (s-1M-1; hydroxyphenylhydantoin) / 0.90 / 24
D-Hydantoinase / F159S / 11.3u / 3.3 to 10 (s-1M-1; hydroxyphenylhydantoin) / 0.66 / 24
D-Hydantoinase / F159I / 11.3u / 3.3 to 13 (s-1M-1; hydroxyphenylhydantoin) / 0.81 / 24
Alicyclobacillus acidocaldarius esterase 2 / M211S / 9.0v / 52 to 107 (s-1 M-1, pNP-decanoate) / 0.43 / 25
Alicyclobacillus acidocaldarius esterase 2 / R215L / 11.5v / 52 to 158 (s-1 M-1, pNP-decanoate) / 0.66 / 25
Alicyclobacillus acidocaldarius esterase 2 / R215L / 11.5v / 15 to 41 (s-1 M-1, pNP-hexanoate) / 0.60 / 25
Subtilisin BPN' / E156S / 10.0w / 0.035 to 0.39 (s-1 M-1, succinyl-Ala-Ala-Pro-Glu-p-nitroanilide) / 1.43 / 26

a Criteria: Only changes in catalytic activity caused by single mutations where the structure of the enzyme is known. Distance from C of mutated amino acid to active site residue unless otherwise specified. Free energy values are calculated at 298 K.

b Distance to closest O of active site Glu372; based on structure of -glucosidase from Bacillus Polymyxa.

c Distance to closest O of active site Asp124.

dDistance to closest O of active site Glu164; based on structure of -glucosidase from Bacillus Polymyxa.

e Distance to anomeric carbon of galactose; based on structure of L. lactis galactokinase.

f Distance to anomeric carbon of galactose; 14.3 Å to closest N of active site Arg 36.

gDistance to O of active site Ser 199; residues are Y149, N237, Ser 170 in protein data bank file 1fm2.

hDistance to closest O of active site Glu504; structure based on human -glucoronidase.

iDistance to N of active site His581.

j Distance to closest O of active site Asp82.

kDistance to O of active site Ser1.

lDistance to P atom of bound substrate analogue diethyl 4-methylbenzylphosphate.

mDistance to P atom of bound substrate analogue diethyl 4-methylbenzylphosphate.

nDistance to N of active site His137; based on methane monooxygenase hydroxylase from Methylococcus capsulatus.

oDistance to N-5 of bound flavine-adenine dinucleotide (FAD).

p Distance to closest O of active site Glu202.

q Distance to O of active site Ser70.

rDistance to O of active site Ser70; based on TEM-1 -Lactamase structure.

s Distance to O of active site Ser70.

t Distance to N of active site His232

uDistance to N of active site His60.

vDistance to O of active site Ser155.

w Distance to Cof P1 residue in bound substrate in protein data bank file 1sua.

References

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