1887

Chapter 31 : Enzyme Engineering: Combining Computational Approaches with Directed Evolution

MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.

Ebook: Choose a downloadable PDF or ePub file. Chapter is a downloadable PDF file. File must be downloaded within 48 hours of purchase

Buy this Chapter
Digital (?) $30.00

Preview this chapter:
Zoom in
Zoomout

Enzyme Engineering: Combining Computational Approaches with Directed Evolution, Page 1 of 2

| /docserver/preview/fulltext/10.1128/9781555816827/9781555815127_Chap31-1.gif /docserver/preview/fulltext/10.1128/9781555816827/9781555815127_Chap31-2.gif

Abstract:

This chapter begins with an overview of the synergy between rational design and screening or selection. The choice of a rational design approach requires consideration of the number of enzyme variants that can be evaluated experimentally. A review of fundamentals of enzyme catalysis from an energy landscape point of view is presented to illustrate why and where rational design could be applied. The bulk of the chapter follows with many examples from the literature of techniques that have worked. Throughout the discussion, mention of basic computational techniques needed to support design is made. It will be seen that it is often quite feasible to adapt an enzyme to many chemical process or organism-engineering needs. Catalytic effects that do not fall well into a thermodynamic viewpoint, such as vibrations or tunneling, are generally smaller, but could also be treated computationally with more development effort. Mutations to proline place a kink in the backbone that can also be stabilizing. The procedure of finding proline mutations can be automated by use of side-chain repacking software. The use of similar sequences to suggest mutations during the evolution of an enzyme is one of the more effective and easiest approaches to stabilization of proteins. Mutations at four positions designed from direct structure comparison helped convert an isocitrate dehydrogenase to an isopropylmalate dehydrogenase, although it was subsequently shown that random mutagenesis was more effective.

Citation: Clark L. 2010. Enzyme Engineering: Combining Computational Approaches with Directed Evolution, p 453-465. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch31

Key Concept Ranking

Aspartate Aminotransferase
0.46829608
Isocitrate Dehydrogenase
0.45637584
Protein Folding
0.45263875
0.46829608
Highlighted Text: Show | Hide
Loading full text...

Full text loading...

Figures

Image of FIGURE 1
FIGURE 1

Possible choices of rational design methods for a given level of screening capacity. The upper triangle gives approximate requirements for structural information in the form of percentage of sequence identity (seqid) of the homology model template to the enzyme sequence. The lower triangle specifies the corresponding screening requirements for each approach Npos, number of positrons targeted.

Citation: Clark L. 2010. Enzyme Engineering: Combining Computational Approaches with Directed Evolution, p 453-465. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch31
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 2
FIGURE 2

Probability of mutating from a given residue type to another during a single-base change. Random mutations are inherently biased on a residue level, and 42% of all residue-to-residue type changes are impossible. Residues are ordered approximately by hydrophobicity. The figure is read from left to right (row index to column index). Example: the probability of mutating from M to I is higher than mutating from I to M.

Citation: Clark L. 2010. Enzyme Engineering: Combining Computational Approaches with Directed Evolution, p 453-465. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch31
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 3
FIGURE 3

A hypothetical energy landscape for an enzyme-catalyzed reaction (black line) compared with the uncatalyzed reaction (gray dotted line). The overall reaction is thermodynamically favorable (ΔG < 0). Notice that, in this case, the catalyzed reaction has only a slightly more favorable activation energy (ΔG) than the uncatalyzed reaction (ΔG) due to strong binding to the substrate (ΔG). E, ezyme; S, substrate; P, product.

Citation: Clark L. 2010. Enzyme Engineering: Combining Computational Approaches with Directed Evolution, p 453-465. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch31
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 4
FIGURE 4

Distribution of mutation effect on the ΔG of protein folding. Data are taken from the Protherm4.0 data set and restricted to single mutations where the protein structure is known ( ). Buried residues are <20% exposed relative to exposure in an alanine-X-alanine tripeptide. Surface residues are defined as having at least 60% relative exposure.

Citation: Clark L. 2010. Enzyme Engineering: Combining Computational Approaches with Directed Evolution, p 453-465. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch31
Permissions and Reprints Request Permissions
Download as Powerpoint

References

/content/book/10.1128/9781555816827.ch31
1. Altamirano, M. M.,, J. M. Blackburn,, C. Aguayo, and, A. R. Fersht. 2002. Retraction. (Altamirano, M. M., J. M. Blackburn, C. Aguayo, and A. R. Fersht. 2000. Directed evolution of new catalytic activity using the alpha/beta-barrel scaffold. Nature 403:617622). Nature 417:468.
2. Amin, N.,, A. D. Liu,, S. Ramer,, W. Aehle,, D. Meijer,, M. Metin,, S. Wong,, P. Gualfetti, and, V. Schellenberger. 2004. Construction of stabilized proteins by combinatorial consensus mutagenesis. Protein Eng. Des. Sel. 17:787793.
3. Arnold, F. H. 2001. Combinatorial and computational challenges for biocatalyst design. Nature 409:253257.
4. Arnold, K.,, L. Bordoli,, J. Kopp, and, T. Schwede. 2006. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22:195201.
5. Ashworth, J.,, J. J. Havranek,, C. M. Duarte,, D. Sussman,, R. J. J. Monnat,, B. L. Stoddard, and, D. Baker, 2006. Computational redesign of endonuclease DNA binding and cleavage specificity. Nature 441:656659.
6. Ballinger, M. D.,, J. Tom, and, J. A. Wells. 1995. Designing subtilisin BPN’ to cleave substrates containing dibasic residues. Biochemistry 34:1331213319.
7. Ballinger, M. D.,, J. Tom, and, J. A. Wells. 1996. Furilisin: a variant of subtilisin BPN’ engineered for cleaving tribasic substrates. Biochemistry 35:1357913585.
8. Bava, K. A.,, M. M. Gromiha,, H. Uedaira,, K. Kitajima, and, A. Sarai. 2004. ProTherm, version 4.0: thermodynamic database for proteins and mutants. Nucleic Acids Res. 32(Database Issue):D120-D121.
9. Beadle, B. M., and, B. K. Shoichet. 2002. Structural bases of stability-function tradeoffs in enzymes. J. Mol. Biol. 321:285296.
10. Benkovic, S. J., and, S. Hammes-Schiffer. 2003. A perspective on enzyme catalysis. Science 301:11961202.
11. Benson, D. E.,, A. E. Haddy, and, H. W. Hellinga. 2002. Converting a maltose receptor into a nascent binuclear copper oxygenase by computational design. Biochemistry 41:32623269.
12. Benson, D. E.,, M. S. Wisz, and, H. W. Hellinga. 2000. Rational design of nascent metalloenzymes. Proc. Natl. Acad. Sci. USA 97:62926297.
13. Bloom, J. D.,, S. T. Labthavikul,, C. R. Otey, and, F. H. Arnold. 2006. Protein stability promotes evolvability. Proc. Natl. Acad. Sci. USA 103:58695874.
14. Boas, F. E., and, P. B. Harbury. 2007. Potential energy functions for protein design. Curr. Opin. Struct. Biol. 17:199204.
15. Bocola, M.,, N. Otte,, K. E. Jaeger,, M. T. Reetz, and, W. Thiel. 2004. Learning from directed evolution: theoretical investigations into cooperative mutations in lipase enantioselectivity. Chembiochem 5:214223.
16. Bocola, M.,, F. Schulz,, F. Leca,, A. Vogel,, M. W. Fraaije, and, M. T. Reetz. 2005. Converting phenylacetone monooxygenase into phenylcyclohexanone monooxygenase by rational design: towards practical Baeyer-Villiger monooxygenases. Adv. Synth. Catal. 347:979986.
17. Boersma, Y. L.,, T. Pijning,, M. S. Bosma,, A. M. van der Sloot,, L. F. Godinho,, M. J. Dröge,, R. T. Winter,, G. van Pouderoyen,, B. W. Dijkstra, and, W. J. Quax. 2008. Loop grafting of Bacillus subtilis lipase a: inversion of enantioselectivity. Chem. Biol. 15:782789.
18. Bolon, D. N., and, S. L. Mayo. 2001. Enzyme-like proteins by computational design. Proc. Natl. Acad. Sci. USA 98:1427414279.
19. Bornscheuer, U. T., and, M. Pohl. 2001. Improved biocatalysts by directed evolution and rational protein design. Curr. Opin. Chem. Biol. 5:137143.
20. Cabrita, L. D.,, D. Gilis,, A. L. Robertson,, Y. Dehouck,, M. Rooman, and, S. P. Bottomley. 2007. Enhancing the stability and solubility of TEV protease using in silico design. Protein Sci. 16:23602367.
21. Carter, P.,, B. Nilsson,, J. P. Burnier,, D. Burdick, and, J. A. Wells. 1989. Engineering subtilisin BPN’ for site-specific proteolysis. Proteins 6:240248.
22. Castagnetto, J. M.,, S. W. Hennessy,, V. A. Roberts,, E. D. Getzoff,, J. A. Tainer, and, M. E. Pique. 2002. MDB: the metalloprotein database and browser at the Scripps Research Institute. Nucleic Acids Res. 30:379382.
23. Cedrone, F.,, A. Ménez, and, E. Quéméneur. 2000. Tailoring new enzyme functions by rational redesign. Curr. Opin. Struct. Biol. 10:405410.
24. Chen, R.,, A. Greer, and, A. M. Dean. 1995. A highly active decarboxylating dehydrogenase with rationally inverted coenzyme specificity. Proc. Natl. Acad. Sci. USA 92:1166611670.
25. Chen, R.,, A. Greer, and, A. M. Dean. 1996. Redesigning secondary structure to invert coenzyme specificity in isopropylmalate dehydrogenase. Proc. Natl. Acad. Sci. USA 93:1217112176.
26. Chen, S.,, M. C. Galan,, C. Coltharp, and, S. E. O’Connor. 2006. Redesign of a central enzyme in alkaloid biosynthesis. Chem. Biol. 13:11371141.
27. Cheon, Y. H.,, H. S. Park,, J. H. Kim,, Y. Kim, and, H. S. Kim. 2004. Manipulation of the active site loops of d-hydantoinase, a (beta/alpha)8-barrel protein, for modulation of the substrate specificity. Biochemistry 43:74137420.
28. Chica, R. A.,, N. Doucet, and, J. N. Pelletier. 2005. Semi-rational approaches to engineering enzyme activity: combining the benefits of directed evolution and rational design. Curr. Opin. Biotechnol. 16:378384.
29. Choi, E. J., and, S. L. Mayo. 2006. Generation and analysis of proline mutants in protein G. Protein Eng. Des. Sel. 19:285289.
30. Choudhury, N., and, B. M. Pettitt. 2006. Enthalpy-entropy contributions to the potential of mean force of nanoscopic hydrophobic solutes. J. Phys. Chem. B 110:84598463.
31. Clark, L. A.,, P. A. Boriack-Sjodin,, E. Day,, J. Eldredge,, C. Fitch,, M. Jarpe,, S. Miller,, Y. Li,, K. Simon, and, H. W. van Vlijmen. 2008. An antibody loop replacement design feasibility study and a loop-swapped dimer structure. Protein Eng. Des. Sel. 22:93101.
32. Coldren, C. D.,, H. W. Hellinga, and, J. P. Caradonna. 1997. The rational design and construction of a cuboidal iron-sulfur protein. Proc. Natl. Acad. Sci. USA 94:66356640.
33. Corey, M. J., and, E. Corey. 1996. On the failure of de novo-designed peptides as biocatalysts. Proc. Natl. Acad. Sci. USA 93:1142811434.
34. Craik, C. S.,, C. Largman,, T. Fletcher,, S. Roczniak,, P. J. Barr,, R. Fletterick, and, W. J. Rutter. 1985. Redesigning tryp-sin: alteration of substrate specificity. Science 228:291297.
35. Crameri, A.,, S. A. Raillard,, E. Bermudez, and, W. P. Stemmer. 1998. DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature 391:288291.
36. Dantas, G.,, B. Kuhlman,, D. Callender,, M. Wong, and, D. Baker. 2003. A large scale test of computational protein design: folding and stability of nine completely redesigned globular proteins. J. Mol. Biol. 332:449460.
37. DeGrado, W. F.,, C. M. Summa,, V. Pavone,, F. Nastri, and, A. Lombardi. 1999. De novo design and structural characterization of proteins and metalloproteins. Annu. Rev. Biochem. 68:779819.
38. Degtyarenko, K. N.,, A. C. North, and, J. B. Findlay. 1999. PROMISE: a database of bioinorganic motifs. Nucleic Acids Res. 27:233236.
39. DeLano, W. 2002. The PyMOL molecular graphics system. DeLano Scientific, Palo Alto, CA. http://www.pymol.org.
40. Reference deleted.
41. Desantis, G.,, K. Wong,, B. Farwell,, K. Chatman,, Z. Zhu,, G. Tomlinson,, H. Huang,, X. Tan,, L. Bibbs,, P. Chen,, K. Kretz, and, M. J. Burk. 2003. Creation of a productive, highly enantioselective nitrilase through gene site saturation mutagenesis (GSSM). J. Am. Chem. Soc. 125:1147611477.
42. Ditursi, M. K.,, S. J. Kwon,, P. J. Reeder, and, J. S. Dordick. 2006. Bioinformatics-driven, rational engineering of protein thermostability. Protein Eng. Des. Sel. 19:517524.
43. Doyle, S. A.,, S. Y. Fung, and, D. E. J. Koshland. 2000. Redesigning the substrate specificity of an enzyme: isocitrate dehydrogenase. Biochemistry 39:1434814355.
44. Dwyer, M. A.,, L. L. Looger, and, H. W. Hellinga. 2008. Retraction (Dwyer, M. A., L. L. Looger, and H. W. Hellinga. 2004. Computational design of a biologically active enzyme. Science 304:19671971). Science 319:569.
45. Reference deleted.
46. Eijsink, V. G.,, A. Bjork,, S. Gaseidnes,, R. Sirevag,, B. Synstad,, B. van den Burg, and, G. Vriend. 2004. Rational engineering of enzyme stability. J. Biotechnol. 113:105120.
47. Eisenmesser, E. Z.,, O. Millet,, W. Labeikovsky,, D. M. Korzhnev,, M. Wolf-Watz,, D. A. Bosco,, J. J. Skalicky,, L. E. Kay, and, D. Kern. 2005. Intrinsic dynamics of an enzyme underlies catalysis. Nature 438:117121.
48. Ema, T.,, T. Fujii,, M. Ozaki,, T. Korenaga, and, T. Sakai. 2005. Rational control of enantioselectivity of lipase by site-directed mutagenesis based on the mechanism. Chem. Commun. (Camb.) 4650:46504651.
49. Fenel, F.,, M. Leisola,, J. Jänis, and, O. Turunen. 2004. A de novo designed n-terminal disulphide bridge stabilizes the Trichoderma reesei endo-1,4-beta-xylanase II. J. Biotechnol. 108:137143.
50. Fersht, A. 1998. Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding. W. H. Freeman & Co., New York, NY.
51. Field, M. J. 2002. Simulating enzyme reactions: challenges and perspectives. J. Comp. Chem. 23:4858.
52. Fletcher, M. C.,, A. Kuderova,, M. Cygler, and, J. S. Lee. 1998. Creation of a ribonuclease abzyme through site-directed mutagenesis. Nat. Biotechnol. 16:10651067.
53. Flores, H., and, A. D. Ellington. 2005. A modified consensus approach to mutagenesis inverts the cofactor specificity of Bacillus stearothermophilus lactate dehydrogenase. Protein Eng. Des. Sel. 18:369377.
54. Fox, R.,, A. Roy,, S. Govindarajan,, J. Minshull,, C. Gustafsson,, J. T. Jones, and, R. Emig. 2003. Optimizing the search algorithm for protein engineering by directed evolution. Protein Eng. 16:589597.
55. Fox, R. J.,, S. C. Davis,, E. C. Mundorff,, L. M. Newman,, V. Gavrilovic,, S. K. Ma,, L. M. Chung,, C. Ching,, S. Tam,, S. Muley,, J. Grate,, J. Gruber,, J. C. Whitman,, R. A. Sheldon, and, G. W. Huisman. 2007. Improving catalytic function by ProSAR-driven enzyme evolution. Nat. Biotechnol. 25:338344.
56. Garcia-Viloca, M.,, J. Gao,, M. Karplus, and, D. G. Truhlar. 2004. How enzymes work: analysis by modern rate theory and computer simulations. Science 303:186195.
57. Gocke, D.,, L. Walter,, E. Gauchenova,, G. Kolter,, M. Knoll,, C. L. Berthold,, G. Schneider,, J. Pleiss,, M. Müller, and, M. Pohl. 2008. Rational protein design of ThDP-dependent enzymes—engineering stereoselectivity. Chembiochem 9:406412.
58. Guerois, R.,, J. E. Nielsen, and, L. Serrano. 2002. Predicting changes in the stability of proteins and protein complexes: a study of more than 1000 mutations. J. Mol. Biol. 320:369387.
59. Hay, M.,, J. H. Richards, and, Y. Lu. 1996. Construction and characterization of an azurin analog for the purple copper site in cytochrome c oxidase. Proc. Natl. Acad. Sci. USA 93:461464.
60. Hellinga, H. W., and, F. M. Richards. 1991. Construction of new ligand binding sites in proteins of known structure. I. Computer-aided modeling of sites with predefined geometry. J. Mol. Biol. 222:763785.
61. Reference deleted.
62. Hill, C. M.,, W. S. Li,, J. B. Thoden,, H. M. Holden, and, F. M. Raushel. 2003. Enhanced degradation of chemical warfare agents through molecular engineering of the phosphotriesterase active site. J. Am. Chem. Soc. 125:89908991.
63. Hilvert, D. 2000. Critical analysis of antibody catalysis. Annu. Rev. Biochem. 69:751793.
64. Höst, G.,, L. G. Mårtensson, and, B. H. Jonsson. 2006. Redesign of human carbonic anhydrase II for increased esterase activity and specificity towards esters with long acyl chains. Biochim. Biophys. Acta 1764:16011606.
65. Hu, X.,, H. Wang,, H. Ke, and, B. Kuhlman. 2007. High-resolution design of a protein loop. Proc. Natl. Acad. Sci. USA 104:1766817673.
66. Jeong, M. Y.,, S. Kim,, C. W. Yun,, Y. J. Choi, and, S. G. Cho, 2007. Engineering a de novo internal disulfide bridge to improve the thermal stability of xylanase from Bacillus stearothermophilus no. 236. J. Biotechnol. 127:300309.
67. Jiang, L.,, E. A. Althoff,, F. R. Clemente,, L. Doyle,, D. Röthlisberger,, A. Zanghellini,, J. L. Gallaher,, J. L. Betker,, F. Tanaka,, C. F. Barbas III,, D. Hilvert,, K. N. Houk,, B. L. Stoddard, and, D. Baker. 2008. De novo computational design of retro-aldol enzymes. Science 319:13871391.
68. Jorgensen, W. L. 1989. Free energy calculations: a breakthrough for modeling organic chemistry in solution. Acc. Chem. Res. 22:184189.
69. Kabashima, T.,, Y. Li,, N. Kanada,, K. Ito, and, T. Yoshimoto. 2001. Enhancement of the thermal stability of pyroglutamyl peptidase I by introduction of an intersubunit disulfide bond. Biochim. Biophys. Acta 1547:214220.
70. Kagami, O.,, K. Shindo,, A. Kyojima,, K. Takeda,, H. Ikenaga,, K. Furukawa, and, N. Misawa. 2008. Protein engineering on biphenyl dioxygenase for conferring activity to convert 7-hydroxyflavone and 5,7-dihydroxyflavone (chrysin). J. Biosci. Bioeng. 106:121127.
71. Kaplan, J., and, W. F. DeGrado. 2004. De novo design of catalytic proteins. Proc. Natl. Acad. Sci. USA 101:1156611570.
72. Karimäki, J.,, T. Parkkinen,, H. Santa,, O. Pastinen,, M. Leisola,, J. Rouvinen, and, O. Turunen. 2004. Engineering the substrate specificity of xylose isomerase. Protein Eng. Des. Sel. 17:861869.
73. Ko, J. H.,, W. H. Jang,, E. K. Kim,, H. B. Lee,, K. D. Park,, J. H. Chung, and, O. J. Yoo. 1996. Enhancement of thermostability and catalytic efficiency of AprP, an alkaline protease from Pseudomonas sp., by the introduction of a disulfide bond. Biochem. Biophys. Res. Commun. 221:631635.
74. Koch, W., and, M. C. Holthausen. 2000. A Chemist’s Guide to Density Functional Theory. John Wiley & Sons, Inc., Hoboken, NJ.
75. Koga, Y.,, K. Kato,, H. Nakano, and, T. Yamane. 2003. Inverting enantioselectivity of Burkholderia cepacia KWI-56 lipase by combinatorial mutation and high-throughput screening using single-molecule PCR and in vitro expression. J. Mol. Biol. 331:585592.
76. Korkegian, A.,, M. E. Black,, D. Baker, and, B. L. Stoddard. 2005. Computational thermostabilization of an enzyme. Science 308:857860.
77. Kristan, K.,, J. Stojan,, J. Adamski, and, T. L. Rizner. 2007. Rational design of novel mutants of fungal 17beta-hydroxysteroid dehydrogenase. J. Biotechnol. 129:12330.
78. Larson, S. M.,, A. A. Di Nardo, and, A. R. Davidson. 2000. Analysis of covariation in an SH3 domain sequence alignment: applications in tertiary contact prediction and the design of compensating hydrophobic core substitutions. J. Mol. Biol. 303:433446.
79. Lassila, J. K.,, J. R. Keeffe,, P. Oelschlaeger, and, S. L. Mayo. 2005. Computationally designed variants of Escherichia coli chorismate mutase show altered catalytic activity. Protein Eng. Des. Sel. 18:161163.
80. Liebeton, K.,, A. Zonta,, K. Schimossek,, M. Nardini,, D. Lang,, B. W. Dijkstra,, M. T. Reetz, and, K. E. Jaeger. 2000. Directed evolution of an enantioselective lipase. Chem. Biol. 7:709718.
81. Luetz, S.,, L. Giver, and, J. Lalonde. 2008. Engineered enzymes for chemical production. Biotechnol. Bioeng. 101:647653.
82. Ma, H., and, T. M. Penning. 1999. Conversion of mammalian 3alpha-hydroxysteroid dehydrogenase to 20alpha-hydroxys-teroid dehydrogenase using loop chimeras: changing specificity from androgens to progestins. Proc. Natl. Acad. Sci. USA 96:1116111166.
83. Magnusson, A. O.,, J. C. Rotticci-Mulder,, A. Santagostino, and, K. Hult. 2005. Creating space for large secondary alcohols by rational redesign of Candida antarctica lipase B. Chembiochem 6:10511056.
84. Masgrau, L.,, A. Roujeinikova,, L. O. Johannissen,, P. Hothi,, J. Basran,, K. E. Ranaghan,, A. J. Mulholland,, M. J. Sutcliffe,, N. S. Scrutton, and, D. Leys. 2006. Atomic description of an enzyme reaction dominated by proton tunneling. Science 312:237241.
85. Matthews, B. W.,, H. Nicholson, and, W. J. Becktel. 1987. Enhanced protein thermostability from site-directed mutations that decrease the entropy of unfolding. Proc. Natl. Acad. Sci. USA 84:66636667.
86. May, O.,, P. T. Nguyen, and, F. H. Arnold. 2000. Inverting enantioselectivity by directed evolution of hydantoinase for improved production of l-methionine. Nat. Biotechnol. 18:317320.
87. Morley, K. L., and, R. J. Kazlauskas. 2005. Improving enzyme properties: when are closer mutations better? Trends Biotechnol. 23:231237.
88. Ness, J. E.,, S. Kim,, A. Gottman,, R. Pak,, A. Krebber,, T. V. Borchert,, S. Govindarajan,, E. C. Mundorff, and, J. Minshull. 2002. Synthetic shuffling expands functional protein diversity by allowing amino acids to recombine independently. Nat. Biotechnol. 20:12511255.
89. Onuffer, J. J., and, J. F. Kirsch. 1995. Redesign of the substrate specificity of Escherichia coli aspartate aminotransferase to that of Escherichia coli tyrosine aminotransferase by homology modeling and site-directed mutagenesis. Protein Sci. 4:17501757.
90. Park, H. S.,, S. H. Nam,, J. K. Lee,, C. N. Yoon,, B. Mannervik,, S. J. Benkovic, and, H. S. Kim. 2006. Design and evolution of new catalytic activity with an existing protein scaffold. Science 311:535538.
91. Park, S.,, K. L. Morley,, G. P. Horsman,, M. Holmquist,, K. Hult, and, R. J. Kazlauskas. 2005. Focusing mutations into the P. fluorescens esterase binding site increases enantioselectivity more effectively than distant mutations. Chem. Biol. 12:4554.
92. Penning, T. M., and, J. M. Jez. 2001. Enzyme redesign. Chem. Rev. 101:30273046.
93. Pinto, A. L.,, H. W. Hellinga, and, J. P. Caradonna. 1997. Construction of a catalytically active iron superoxide dismutase by rational protein design. Proc. Natl. Acad. Sci. USA 94:55625567.
94. Pollard, D. J., and, J. M. Woodley. 2007. Biocatalysis for pharmaceutical intermediates: the future is now. Trends Biotechnol. 25:6673.
95. Reetz, M. T. 2004. Changing the enantioselectivity of enzymes by directed evolution. Methods Enzymol. 388:238256.
96. Reetz, M. T., and, J. D. Carballeira. 2007. Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes. Nat. Protoc. 2:891903.
97. Reetz, M. T.,, S. Wilensek,, D. Zha, and, K. E. Jaeger. 2001. Directed evolution of an enantioselective enzyme through combinatorial multiple-cassette mutagenesis. Angew. Chem. Int. Ed. Engl. 40:35893591.
98. Rheinnecker, M.,, G. Baker,, J. Eder, and, A. R. Fersht. 1993. Engineering a novel specificity in subtilisin BPN’. Biochemistry 32:11991203.
99. Röthlisberger, D.,, O. Khersonsky,, A. M. Wollacott,, L. Jiang,, J. Dechancie,, J. Betker,, J. L. Gallaher,, E. A. Althoff,, A. Zanghellini,, O. Dym,, S. Albeck,, K. N. Houk,, D. S. Tawfik, and, D. Baker. 2008. Kemp elimination catalysts by computational enzyme design. Nature 453:190195.
100. Rui, L.,, L. Cao,, W. Chen,, K. F. Reardon, and, T. K. Wood. 2004. Active site engineering of the epoxide hydrolase from Agrobacterium radiobacter AD1 to enhance aerobic mineralization of cis-1,2-dichloroethylene in cells expressing an evolved toluene ortho-monooxygenase. J. Biol. Chem. 279:4681046817.
101. Russ, W. P., and, R. Ranganathan. 2002. Knowledge-based potential functions in protein design. Curr. Opin. Struct. Biol. 12:447452.
102. Sali, A., and, T. L. Blundell. 1993. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234:779815.
103. Schmitzer, A. R.,, F. Lépine, and, J. N. Pelletier. 2004. Combinatorial exploration of the catalytic site of a drug-resistant dihydrofolate reductase: creating alternative functional configurations. Protein Eng. Des. Sel. 17:809819.
104. Scrutton, N. S.,, A. Berry, and, R. N. Perham. 1990. Redesign of the coenzyme specificity of a dehydrogenase by protein engineering. Nature 343:3843.
105. Seelig, B., and, J. W. Szostak. 2007. Selection and evolution of enzymes from a partially randomized non-catalytic scaffold. Nature 448:828831.
106. Shoichet, B. K.,, W. A. Baase,, R. Kuroki, and, B. W. Matthews. 1995. A relationship between protein stability and protein function. Proc. Natl. Acad. Sci. USA 92:452456.
107. Sigman, J. A.,, B. C. Kwok, and, Y. Lu. 2000. From myoglobin to hemecopper oxidase: design and engineering of a CuB center into sperm whale myoglobin. J. Am. Chem. Soc. 122:81928196.
108. Steipe, B.,, B. Schiller,, A. Plückthun, and, S. Steinbacher. 1994. Sequence statistics reliably predict stabilizing mutations in a protein domain. J. Mol. Biol. 240:188192.
109. Takagi, H.,, T. Takahashi,, H. Momose,, M. Inouye,, Y. Maeda,, H. Matsuzawa, and, T. Ohta. 1990. Enhancement of the thermostability of subtilisin e by introduction of a disulfide bond engineered on the basis of structural comparison with a thermophilic serine protease. J. Biol. Chem. 265:68746878.
110. Tang, L.,, D. E. T. Pazmino,, M. W. Fraaije,, R. M. D. Jong,, B. W. Dijkstra, and, D. B. Janssen. 2005. Improved catalytic properties of halohydrin dehalogenase by modification of the halide-binding site. Biochemistry 44:66096618.
111. van den Burg, B.,, B. W. Dijkstra,, B. van der Vinne,, B. K. Stulp,, V. G. Eijsink, and, G. Venema. 1993. Introduction of disulfide bonds into Bacillus subtilis neutral protease. Protein Eng. 6:521527.
112. van den Heuvel, R. H.,, M. W. Fraaije,, M. Ferrer,, A. Mattevi, and, W. J. van Berkel. 2000. Inversion of stereospecificity of vanillylalcohol oxidase. Proc. Natl. Acad. Sci. USA 97:94559460.
113. Vázquez-Figueroa, E.,, J. Chaparro-Riggers, and, A. S. Bommarius. 2007. Development of a thermostable glucose dehydrogenase by a structure-guided consensus concept. Chembiochem 8:22952301.
114. Vazquez-Figueroa, E.,, V. Yeh,, J. M. Broering,, J. F. Chaparro-Riggers, and, A. S. Bommarius. 2008. Thermostable variants constructed via the structure-guided consensus method also show increased stability in salts solutions and homogeneous aqueous-organic media. Protein Eng. Des. Sel. 21:673680.
115. Vieille, C., and, G. J. Zeikus. 2001. Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 65:143.
116. Voigt, C. A.,, C. Martinez,, Z. G. Wang,, S. L. Mayo, and, F. H. Arnold. 2002. Protein building blocks preserved by recombination. Nat. Struct. Biol. 9:553558.
117. Wakarchuk, W. W.,, W. L. Sung,, R. L. Campbell,, A. Cunningham,, D. C. Watson, and, M. Yaguchi. 1994. Thermostabilization of the Bacillus circulans xylanase by the introduction of disulfide bonds. Protein Eng. 7:13791386.
118. Wells, J. A.,, D. B. Powers,, R. R. Bott,, T. P. Graycar, and, D. A. Estell. 1987. Designing substrate specificity by protein engineering of electrostatic interactions. Proc. Natl. Acad. Sci. USA 84:12191223.
119. Wu, R.,, A. S. Reger,, J. Cao,, A. M. Gulick, and, D. Dunaway-Mariano. 2007. Rational redesign of the 4-chlorobenzoate binding site of 4-chlorobenzoate: co-enzyme A ligase for expanded substrate range. Biochemistry 46:1448714499.
120. Yoshikuni, Y.,, T. E. Ferrin, and, J. D. Keasling. 2006. Designed divergent evolution of enzyme function. Nature 440:10781082.
121. Zanghellini, A.,, L. Jiang,, A. M. Wollacott,, G. Cheng,, J. Meiler,, E. A. Althoff,, D. Rothlisberger, and, D. Baker. 2006. New algorithms and an in silico benchmark for computational enzyme design. Protein Sci. 15:27852794.
122. Zhang, X., and, K. N. Houk. 2005. Why enzymes are proficient catalysts: beyond the Pauling paradigm. Acc. Chem. Res. 38:379385.

This is a required field
Please enter a valid email address
Please check the format of the address you have entered.
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error