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Chap 15. Protein Engineering 1. Dissection of the structure and activity of existing proteins 2. Production of novel proteins: enzymes and antibodies Protein Engineering for Altering Enzyme Reaction Specificity 1. 2. 3. 4. Directed evolution Sequence comparison Structure comparison Altering mechanism (Modification of the catalytic machinery for a non-related catalytic activity) 5. Introduction of the whole catalytic machinery 6. Non-catalytic protein template (Introduction of catalytic activity into non-catalytic protein) Berglund, P.; Park, S. Current Organic Chemistry, 2005, 9, 325-336 Evolution of New Enzyme Activity in Nature Divergent evolution (a) Gerlt, J.A.; Babbitt, P.C. Curr. Opin. Chem. Biol., 1998, 2, 607-612. (b) Babbitt, P.C.; Gerlt, J.A. J. Biol. Chem., 1997, 272, 30591-30594. Convergent evolution Todd, A.E.; Orengo, C.A.; Thornton, J.M. Trends Biochem. Sci., 2002, 27, 419-426. 1. Oxydosqualene cyclization produces different steroids according to the site of deprotonation by different enzymes H One mutant of cycloartenol synthase can produce lanosterol H cycloartenol HO - H at C-19 H H H 19 11 H - H at C-8 8 HO O oxydosqualene lanosterol HO H lanosteryl cation H - H at C-11 cycloartenol synthase (EC 5.4.99.8) lanosterol synthase (EC 5.4.99.7) H H parkeol HO H Selection: Lanostrerol is an intermediate of ergosterol that is an essential fungal membrane component Hart, E. A.; Hua, L.; Darr, L. B.; Wilson, W. K.; Pang, J.; Matsuda, S. P. T. J. Am. Chem. Soc., 1999, 121, 9887-9888. 2. Substitutions of several residues interchange the catalytic activity between a desaturase and a hydroxylase hydroxylase OH 12 linoleic acid O OH oleic acid O desaturase HO OH oleate hydroxylase (EC 1.14.13.26) ricinoleic acid O oleate desaturase (EC 1.3.1.35) Six amino acid substitutions converted oleate hydroxylase to a desaturase. Four amino acid substitutions converted oleate desaturase to a hydroxylase. Broun, P.; Shanklin, J.; Whittle, E.; Somerville, C. Science, 1998, 282, 1315-1317. 2. A rationally designed mutant of glutathione transferase A1-1 shows Michael addition activity glutathione transferase (GST, EC 2.5.1.18) 4 aa substitutions in A1-1 + Cterminus from A4-4 into A1-1 gave 300-fold increase in Michael activity and 10-fold decrease in subst activity. Nilsson, L.; Gustafsson, A.; Mannervik, B. Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 9408-9412. 3. The muconate lactonizing enzyme subgroup catalyze three different reactions through deprotonation of an α-proton muconate lactonizing enzymes (EC 5.5.1.1) CO2 O O O O O H N H O O O HN Ala AEE Ala O O H O O H H O H O O O H O O O H HO HO O2C H O HN Ala O MLE II OSBS CO2 CO2 O O O O H O O O O2C O O2C O racemization Single-site mutants of the L-Ala-D/L-Glu epimerase and The Ring opening muconate lactonizing enzyme II show the osuccinyl-benzoatesynthase activity b-elimination Schmidt, D. M. Z.; Mundorff, E. C.; Dojka, M.; Bermudez, E.; Ness, J. E.; Govindarajan, S. G.; Babbitt, P. C.; Minshull, J.; Gerlt, J. A. Biochemistry, 2003, 42, 8387-8393. 3. 2-Enoyl-CoA hydratase (Crotonase) and 4-CBA-CoA dehalogenase have similar active sites but catalyze different reactions O crotonase H2O CoAS O OH Michael addition CoAS Nucleophilic substitution O O 4-CBA-CoA dehalogenease + Cl CoAS CoAS H2O Cl OH Active site overlapping resulted in a seven-residue substitution in 4-CBA-CoA dehalogenase which showed crotonase activity. Xiang, H.; Luo, L.; Taylor, K. L.; Dunaway-Mariano, D. Biochemistry, 1999, 38, 7638-7652. 3. The reaction specificity of alanine racemase altered into that of an aminotransferase by a double active-site mutation alanine racemase H2N H2N CO2 L-alanine CO2 O D-alanine D-amino acid aminotranferase CO2 pyruvate Arg219Glu resulted in 103-fold decrease in racemase activity and a 5.4-fold increase in transaminase activity. Tyr265Ala eliminated racemase activity. (a) Watanabe, A.; Yoshimura, T.; Mikami, B.; Hayashi, H.; Kagamiyama, H.; Esaki, N. J. Biol. Chem., 2002, 277, 19166-19172. (b) Yow, G.-Y.; Watanabe, A.; Yoshimura, T.; Esaki, N. J. Mol. Catal. B: Enzym., 2003, 23, 311-319. 3. Conversion of ala racemase into an aldolase with a single-point mutation Alanine racemase from Geobacillus stearothermophilus (EC 5.1.1.1) L-threonine aldolase (EC 4.1.2.5) base H2N CO2 O O P O O OH D-(2R,3S)-phenylserine CO2 HO H N N H H O CH3 b-phenylserine-PLP aldimine O H2N CO2 + glycine retro-aldol reaction Tyr265Ala mutant: 3 103 fold reduced racemase activity and 2.3 105 fold increased aldolase activity with D-(2R,3S)-phenylserine, compared to the wild type enzyme. Highly enantioselective. Seebeck, F.P.; Hilvert, D. J. Am. Chem. Soc., 2003, 125, 10158-10159. H 4. Hydrolysis of nitriles by a papain mutant R2 R2 H N CN R1 S Enzyme R1 a thioimidate Wild type nitrile hydratase (EC 4.2.1.84) nitrilases (EC 3.5.5.X) papain (EC 3.4.22.2) NH H N X Papain Gln19Glu O2C Glu19 H R2 H N N H R2 S Enzyme R1 O H N NH2 R2 O H N R1 H2O Dufour, E.; Storer, A.C.; Ménard, R. Biochemistry, 1995, 34, 16382-16388. OH + NH3 R1 4. Aldol addition activity of C. antarctica lipase B O O R H R C. antarctica lipase B Ser105Ala HO Ala105 CH3 H O H R Asp187 O H N N O H H Oxyanion hole R His224 Substrate docking 250 ps dynamics O H 3-methyl-butanal Asp187 Gln106 Ala105 Thr40 His224 Critical H-bond distance Trp104 (a) Branneby, C.; Carlqvist, P.; Magnusson, A.; Hult, K.; Brinck, T.; Berglund, P. J. Am. Chem. Soc., 2003, 125, 874-875. (b) Branneby, C.; Carlqvist, P.; Hult, K.; Brinck, T.; Berglund, P. J. Mol. Catal. B: Enzym., 2004, 31, 123-128. 5. Conversion of cyclophilin into a proline-specific endopeptidase (hydrolase) Cyclophilin (EC 5.2.1.8) O cyclophilin N OH O O N OH R O R H2N cis X-Pro Cis-trans isomerization NH2 trans X-Pro 1. Several candidate sites for the serine evaluated 2. Rest of triad introduced in best serine mutant New hydrolytic activity was 8 108-fold over the uncatalyzed reaction Quéméneur, E.; Moutiez, M.; Charbonnier, J.-B.; Ménez, A. Nature, 1998, 391, 301-304. 6. Introduction of catalytic activity into non-catalytic proteins Scytalone dehydratase activity (EC 4.2.1.94) into nuclear transport factor 2 OH O HO OH OH - H2O OH a mutant of NTF2 HO 8 mutations caused 150-fold activity improvement Nixon, A. E.; Firestine, S. M.; Salinas, F. G.; Benkovic, S. J. Proc. Natl. Acad. Sci. U.S.A., 1999, 96, 3568-3571. 6. Introduction of catalytic activity into non-catalytic proteins Triose phosphate isomerase (TIM, EC 5.3.1.1) activity into ribose binding protein The most active mutant (NovoTim 1.2) of RBP contains 13-amino acid substitutions and shows 105-fold rate improvement over the uncatalyzed reaction. (a) Looger, L. L.; Dwyer, M. A.; Smith, J. J.; Hellinga, H. W. Nature, 2003, 423, 185-190. (b) Dwyer, M. A.; Looger, L. L.; Hellinga, H. W. Science, 2004, 304, 1967-1971