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.Directed evolution 2.Sequence comparison 3.Structure comparison 4.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,

Evolution of New Enzyme Activity in Nature Divergent evolutionConvergent evolution (a) Gerlt, J.A.; Babbitt, P.C. Curr. Opin. Chem. Biol., 1998, 2, (b) Babbitt, P.C.; Gerlt, J.A. J. Biol. Chem., 1997, 272, Todd, A.E.; Orengo, C.A.; Thornton, J.M. Trends Biochem. Sci., 2002, 27,

1. Oxydosqualene cyclization produces different steroids according to the site of deprotonation by different enzymes One mutant of cycloartenol synthase can produce lanosterol cycloartenol synthase (EC ) lanosterol synthase (EC ) 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,

2. Substitutions of several residues interchange the catalytic activity between a desaturase and a hydroxylase Broun, P.; Shanklin, J.; Whittle, E.; Somerville, C. Science, 1998, 282, oleate desaturase (EC ) oleate hydroxylase (EC ) Six amino acid substitutions converted oleate hydroxylase to a desaturase. Four amino acid substitutions converted oleate desaturase to a hydroxylase.

2. A rationally designed mutant of glutathione transferase A1-1 shows Michael addition activity Nilsson, L.; Gustafsson, A.; Mannervik, B. Proc. Natl. Acad. Sci. U.S.A., 2000, 97, glutathione transferase (GST, EC ) 4 aa substitutions in A1-1 + C- terminus from A4-4 into A1-1 gave 300-fold increase in Michael activity and 10-fold decrease in subst activity.

3. The muconate lactonizing enzyme subgroup catalyze three different reactions through deprotonation of an α-proton Single-site mutants of the L-Ala-D/L-Glu epimerase and The muconate lactonizing enzyme II show the o- succinyl-benzoate- synthase activity 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, racemization  -elimination Ring opening muconate lactonizing enzymes (EC )

3. 2-Enoyl-CoA hydratase (Crotonase) and 4-CBA-CoA dehalogenase have similar active sites but catalyze different reactions Xiang, H.; Luo, L.; Taylor, K. L.; Dunaway-Mariano, D. Biochemistry, 1999, 38, Michael addition Nucleophilic substitution Active site overlapping resulted in a seven-residue substitution in 4-CBA-CoA dehalogenase which showed crotonase activity.

3. The reaction specificity of alanine racemase altered into that of an aminotransferase by a double active-site mutation (a) Watanabe, A.; Yoshimura, T.; Mikami, B.; Hayashi, H.; Kagamiyama, H.; Esaki, N. J. Biol. Chem., 2002, 277, (b) Yow, G.-Y.; Watanabe, A.; Yoshimura, T.; Esaki, N. J. Mol. Catal. B: Enzym., 2003, 23, Arg219Glu resulted in fold decrease in racemase activity and a 5.4-fold increase in transaminase activity. Tyr265Ala eliminated racemase activity.

3. Conversion of ala racemase into an aldolase with a single-point mutation Alanine racemase from Geobacillus stearothermophilus (EC ) L-threonine aldolase (EC ) Tyr265Ala mutant: 3  10 3 fold reduced racemase activity and 2.3  10 5 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, retro-aldol reaction

4. Hydrolysis of nitriles by a papain mutant nitrile hydratase (EC ) nitrilases (EC X) papain (EC ) Dufour, E.; Storer, A.C.; Ménard, R. Biochemistry, 1995, 34,

4. Aldol addition activity of C. antarctica lipase B (a)Branneby, C.; Carlqvist, P.; Magnusson, A.; Hult, K.; Brinck, T.; Berglund, P. J. Am. Chem. Soc., 2003, 125, (b) Branneby, C.; Carlqvist, P.; Hult, K.; Brinck, T.; Berglund, P. J. Mol. Catal. B: Enzym., 2004, 31, Asp187 His224 Ala105 Trp104 Thr40 Gln106 Substrate docking 250 ps dynamics 3-methyl-butanal Critical H-bond distance

5. Conversion of cyclophilin into a proline-specific endopeptidase (hydrolase) Quéméneur, E.; Moutiez, M.; Charbonnier, J.-B.; Ménez, A. Nature, 1998, 391, Cyclophilin (EC ) 1.Several candidate sites for the serine evaluated 2.Rest of triad introduced in best serine mutant New hydrolytic activity was 8  fold over the uncatalyzed reaction Cis-trans isomerization

6. Introduction of catalytic activity into non-catalytic proteins Scytalone dehydratase activity (EC ) into nuclear transport factor 2 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,

6. Introduction of catalytic activity into non-catalytic proteins (a) Looger, L. L.; Dwyer, M. A.; Smith, J. J.; Hellinga, H. W. Nature, 2003, 423, (b) Dwyer, M. A.; Looger, L. L.; Hellinga, H. W. Science, 2004, 304, The most active mutant (NovoTim 1.2) of RBP contains 13-amino acid substitutions and shows fold rate improvement over the uncatalyzed reaction. Triose phosphate isomerase (TIM, EC ) activity into ribose binding protein