Switch of substrate specificity of hyperthermophilic acylaminoacyl peptidase by combination of protein and solvent engineering

Abstract

The inherent evolvability of promiscuous enzymes endows them with great potential to be artificially evolved for novel functions. Previously, we succeeded in transforming a promiscuous acylaminoacyl peptidase (apAAP) from the hyperthermophilic archaeon Aeropyrum pernix K1 into a specific carboxylesterase by making a single mutation. In order to fulfill the urgent requirement of thermostable lipolytic enzymes, in this paper we describe how the substrate preference of apAAP can be further changed from p-nitrophenyl caprylate (pNP-C8) to p-nitrophenyl laurate (pNP-C12) by protein and solvent engineering. After one round of directed evolution and subsequent saturation mutagenesis at selected residues in the active site, three variants with enhanced activity towards pNP-C12 were identified. Additionally, a combined mutant W474V/F488G/R526V/T560W was generated, which had the highest catalytic efficiency (k_cat/K_m) for pNP-C12, about 71-fold higher than the wild type. Its activity was further increased by solvent engineering, resulting in an activity enhancement of 280-fold compared with the wild type in the presence of 30% DMSO. The structural basis for the improved activity was studied by substrate docking and molecular dynamics simulation. It was revealed that W474V and F488G mutations caused a significant change in the geometry of the active center, which may facilitate binding and subsequent hydrolysis of bulky substrates. In conclusion, the combination of protein and solvent engineering may be an effective approach to improve the activities of promiscuous enzymes and could be used to create naturally rare hyperthermophilic enzymes.

Key words： acylaminoacyl peptidase; esterase; substrate specificity; protein engineering; solvent engineering

References

[1] Aharoni, A., Gaidukov, L., Khersonsky, O., Mc, Q.G.S., Roodveldt, C., and Tawfik, D.S. (2005). The 'evolvability' of promiscuous protein functions. Nat Genet 37, 73-76 .
[2] Bartlam, M., Wang, G., Yang, H., Gao, R., Zhao, X., Xie, G., Cao, S., Feng, Y., and Rao, Z. (2004). Crystal structure of an acylpeptide hydrolase/esterase from Aeropyrum pernix K1. Structure 12, 1481-1488 10.1016/j.str.2004.05.019.
[3] Gao, R., Feng, Y., Ishikawa, K., Ishida, H., Ando, S., Kosugi, Y., and Cao, S. (2003). Cloning, purification and properties of a hyperthermophilic esterase from archaeon Aeropyrum pernix K1. J Mol Catal, B Enzym 24-25, 1-8 10.1016/S1381-1177(03)00064-X.
[4] Ho, B.K., and Gruswitz, F. (2008). HOLLOW: generating accurate representations of channel and interior surfaces in molecular structures. BMC Struct Biol 8, 4910.1186/1472-6807-8-49.
[5] Houde, A., Kademi, A., and Leblanc, D. (2004). Lipases and their industrial applications: an overview. Appl Biochem Biotechnol 118, 155-170 10.1385/ABAB:118:1-3:155.
[6] Hult, K., and Berglund, P. (2007). Enzyme promiscuity: mechanism and applications. Trends Biotechnol 25, 231-238 10.1016/j.tibtech.2007.03.002.
[7] Imanaka, T., and Atomi, H. (2002). Catalyzing "hot" reactions: enzymes from hyperthermophilic Archaea. Chem Rec 2, 149-163 10.1002/tcr.10023.
[8] Laskowski, R.A., MacArthur, M.W., Moss, D.S., and Thornton, J.M. (1993). PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Cryst 26, 283-291 10.1107/S0021889892009944.
[9] Luetz, S., Giver, L., and Lalonde, J. (2008). Engineered enzymes for chemical production. Biotechnol Bioeng 101, 647-653 10.1002/bit.22077.
[10] Luthy, R., Bowie, J.U., and Eisenberg, D. (1992). Assessment of protein models with three-dimensional profiles. Nature 356, 83-85 10.1038/356083a0.
[11] Madigan, M.T., and Martinko, J.M. (2006). Brock Biology of Microorganisms, 11th ed. Beijing: Science Press.
[12] Mandrich, L., Manco, G., Rossi, M., Floris, E., Jansen-van den Bosch, T., Smit, G., and Wouters, J.A. (2006). Alicyclobacillus acidocaldarius thermophilic esterase EST2's activity in milk and cheese models. Appl Environ Microbiol 72, 3191-3197 10.1128/AEM.72.5.3191-3197.2006.
[13] Marti-Renom, M.A., Stuart, A.C., Fiser, A., Sanchez, R., Melo, F., and Sali, A. (2000). Comparative protein structure modeling of genes and genomes. Annu Rev Biophys Biomol Struct 29, 291-325 .
[14] Martínez, L., Andrade, R., Birgin, E.G., and Martinez, J.M. (2009). PACKMOL: a package for building initial configurations for molecular dynamics simulations. J Comput Chem 30, 2157-2164 10.1002/jcc.21224.
[15] Morley, K.L., and Kazlauskas, R.J. (2005). Improving enzyme properties: when are closer mutations better? Trends Biotechnol 23, 231-237 10.1016/j.tibtech.2005.03.005.
[16] Niehaus, F., Bertoldo, C., Kahler, M., and Antranikian, G. (1999). Extremophiles as a source of novel enzymes for industrial application. Appl Microbiol Biotechnol 51, 711-729 10.1007/s002530051456.
[17] O'Brien, P.J., and Herschlag, D. (1999). Catalytic promiscuity and the evolution of new enzymatic activities. Chem Biol 6, R91-R105 10.1016/S1074-5521(99)80033-7.
[18] Paramesvaran, J., Hibbert, E.G., Russell, A.J., and Dalby, P.A. (2009). Distributions of enzyme residues yielding mutants with improved substrate specificities from two different directed evolution strategies. Protein Eng Des Sel 22, 401-411 10.1093/protein/gzp020.
[19] Rui, L., Kwon, Y.M., Fishman, A., Reardon, K.F., and Wood, T.K. (2004). Saturation mutagenesis of toluene ortho-monooxygenase of Burkholderia cepacia G4 for Enhanced 1-naphthol synthesis and chloroform degradation. Appl Environ Microbiol 70, 3246-3252 10.1128/AEM.70.6.3246-3252.2004.
[20] Salameh, M., and Wiegel, J. (2007). Lipases from extremophiles and potential for industrial applications. Adv Appl Microbiol 61, 253-283 10.1016/S0065-2164(06)61007-1.
[21] Schuttelkopf, A.W., and van Aalten, D.M. (2004). PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr D Biol Crystallogr 60, 1355-1363 10.1107/S0907444904011679.
[22] Soares, C.M., Teixeira, V.H., and Baptista, A.M. (2003). Protein structure and dynamics in nonaqueous solvents: insights from molecular dynamics simulation studies. Biophys J 84, 1628-1641 10.1016/S0006-3495(03)74972-8.
[23] Stahl, M., Jeppsson-Wistrand, U., Mansson, M.O., and Mosbach, K. (1991). Induced stereo- and substrate selectivity of bioimprinted α-chymotrypsin in anhydrous organic media. J Am Chem Soc 113, 9366-9368 10.1021/ja00024a051.
[24] Tawaki, S., and Klibanov, A.M. (1992). Inversion of enzyme enantioselectivity mediated by the solvent. J Am Chem Soc 114, 1882-1884 10.1021/ja00031a054.
[25] Van Der Spoel, D., Lindahl, E., Hess, B., Groenhof, G., Mark, A.E., and Berendsen, H.J. (2005). GROMACS: fast, flexible, and free. J Comput Chem 26, 1701-1718 10.1002/jcc.20291.
[26] Vartanian, J.P., Henry, M., and Wain-Hobson, S. (2001). Simulating pseudogene evolution in vitro: determining the true number of mutations in a lineage. Proc Natl Acad Sci USA 98, 13172-13176 10.1073/pnas.221334898.
[27] Verma, M.L., Azmi, W., and Kanwar, S.S. (2008). Microbial lipases: at the interface of aqueous and non-aqueous media. A review. Acta Microbiol Immunol Hung 55, 265-294 10.1556/AMicr.55.2008.3.1.
[28] Wang, Q., Yang, G., Liu, Y., and Feng, Y. (2006). Discrimination of esterase and peptidase activities of acylaminoacyl peptidase from hyperthermophilic Aeropyrum pernix K1 by a single mutation. J Biol Chem 281, 18618-18625 10.1074/jbc.M601015200.
[29] Wescott, C.R., and Klibanov, A.M. (1993). Solvent variation inverts substrate specificity of an enzyme. J Am Chem Soc 115, 1629-1631 10.1021/ja00058a002.
[30] Yang, G., Bai, A., Gao, L., Zhang, Z., Zheng, B., and Feng, Y. (2009). Glu88 in the non-catalytic domain of acylpeptide hydrolase plays dual roles: charge neutralization for enzymatic activity and formation of salt bridge for thermodynamic stability. Biochim Biophys Acta 1794, 94-102 .
[31] Yang, L., Dordick, J.S., and Garde, S. (2004). Hydration of enzyme in nonaqueous media is consistent with solvent dependence of its activity. Biophys J 87, 812-821 10.1529/biophysj.104.041269.
[32] Zaks, A., and Klibanov, A.M. (1985). Enzyme-catalyzed processes in organic solvents. Proc Natl Acad Sci USA 82, 3192-3196 10.1073/pnas.82.10.3192.
[33] Zheng, L., Baumann, U., and Reymond, J.L. (2004). An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic Acids Res 32, e11510.1093/nar/gnh110.