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Improving the thermostability of methyl parathion hydrolase from Ochrobactrum sp. M231 using a computationally aided method

  • Applied genetics and molecular biotechnology
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Abstract

Good protein thermostability is very important for the protein application. In this report, we propose a strategy which contained a prediction method to select residues related to protein thermal stability, but not related to protein function, and an experiment method to screen the mutants with enhanced thermostability. The prediction strategy was based on the calculated site evolutionary entropy and unfolding free energy difference between the mutant and wild-type (WT) methyl parathion hydrolase enzyme from Ochrobactrum sp. M231 [Ochr-methyl parathion hydrolase (MPH)]. As a result, seven amino acid sites within Ochr-MPH were selected and used to construct seven saturation mutagenesis libraries. The results of screening these libraries indicated that six sites could result in mutated enzymes exhibiting better thermal stability than the WT enzyme. A stepwise evolutionary approach was designed to combine these selected mutants and a mutant with four point mutations (S274Q/T183E/K197L/S192M) was selected. The T m and T 50 of the mutant enzyme were 11.7 and 10.2 °C higher, respectively, than that of the WT enzyme. The success of this design methodology for Ochr-MPH suggests that it was an efficient strategy for enhancing protein thermostability and suitable for protein engineering.

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References

  • Benedix A, Becker CM, de Groot BL, Caflisch A, Bockmann RA (2009) Predicting free energy changes using structural ensembles. Nat Methods 6(1):3–4

    Article  CAS  Google Scholar 

  • Brooks BR, Brooks CL 3rd, Mackerell AD Jr, Nilsson L, Petrella RJ, Roux B, Won Y, Archontis G, Bartels C, Boresch S, Caflisch A, Caves L, Cui Q, Dinner AR, Feig M, Fischer S, Gao J, Hodoscek M, Im W, Kuczera K, Lazaridis T, Ma J, Ovchinnikov V, Paci E, Pastor RW, Post CB, Pu JZ, Schaefer M, Tidor B, Venable RM, Woodcock HL, Wu X, Yang W, York DM, Karplus M (2009) CHARMM: the biomolecular simulation program. J Comput Chem 30(10):1545–614

    Article  CAS  Google Scholar 

  • Capriotti E, Fariselli P, Casadio R (2005) I-Mutant2.0: predicting stability changes upon mutation from the protein sequence or structure. Nucleic Acids Res 33(Web Server issue):W306-10

    Google Scholar 

  • Chen J, Brooks CL 3rd, Khandogin J (2008) Recent advances in implicit solvent-based methods for biomolecular simulations. Curr Opin Struct Biol 18(2):140–8

    Article  CAS  Google Scholar 

  • Choudhury D, Biswas S, Roy S, Dattagupta JK (2010) Improving thermostability of papain through structure-based protein engineering. Protein Eng Des Sel

  • Cui Z, Li S, Fu G (2001) Isolation of methyl parathion-degrading strain M6 and cloning of the methyl parathion hydrolase gene. Appl Environ Microbiol 67(10):4922–5

    Article  CAS  Google Scholar 

  • Dong Y, Bartlam M, Sun L, Zhou Y, Zhang Z, Zhang C, Rao Z, Zhang X (2005) Crystal structure of methyl parathion hydrolase from Pseudomonas sp. WBC-3. J Mol Biol 353(3):655–63

    Article  CAS  Google Scholar 

  • Firth AE, Patrick WM (2005) Statistics of protein library construction. Bioinformatics 21(15):3314–5

    Article  CAS  Google Scholar 

  • Gribenko AV, Patel MM, Liu J, McCallum SA, Wang C, Makhatadze GI (2009) Rational stabilization of enzymes by computational redesign of surface charge-charge interactions. Proc Natl Acad Sci 106(8):2601–2606

    Article  CAS  Google Scholar 

  • Han ZL, Han SY, Zheng SP, Lin Y (2009) Enhancing thermostability of a Rhizomucor miehei lipase by engineering a disulfide bond and displaying on the yeast cell surface. Appl Microbiol Biotechnol 85(1):117–26

    Article  CAS  Google Scholar 

  • Heinzelman P, Snow CD, Wu I, Nguyen C, Villalobos A, Govindarajan S, Minshull J, Arnold FH (2009) A family of thermostable fungal cellulases created by structure-guided recombination. Proc Natl Acad Sci U S A 106(14):5610–5

    Article  CAS  Google Scholar 

  • Jeong MY, Kim S, Yun CW, Choi YJ, Cho SG (2007) Engineering a de novo internal disulfide bridge to improve the thermal stability of xylanase from Bacillus stearothermophilus No. 236. J Biotechnol 127(2):300–9

    Article  CAS  Google Scholar 

  • Kang S, Chen G, Xiao G (2009) Robust prediction of mutation-induced protein stability change by property encoding of amino acids. Protein Eng Des Sel 22(2):75–83

    Article  CAS  Google Scholar 

  • Khan S, Vihinen M (2010) Performance of protein stability predictors. Hum Mutat 31(6):675–84

    Article  CAS  Google Scholar 

  • Korkegian A, Black ME, Baker D, Stoddard BL (2005) Computational thermostabilization of an enzyme. Science 308(5723):857–60

    Article  CAS  Google Scholar 

  • Matsui I, Harata K (2007) Implication for buried polar contacts and ion pairs in hyperthermostable enzymes. FEBS J 274(16):4012–22

    Article  CAS  Google Scholar 

  • McCullum EO, Williams BA, Zhang J, Chaput JC (2010) Random mutagenesis by error-prone PCR. Methods Mol Biol 634:103–9

    Article  CAS  Google Scholar 

  • Parikh MR, Matsumura I (2005) Site-saturation mutagenesis is more efficient than DNA shuffling for the directed evolution of β-fucosidase from β-galactosidase. J Mol Biol 352(3):621–628

    Article  CAS  Google Scholar 

  • Patrick WM, Firth AE (2005) Strategies and computational tools for improving randomized protein libraries. Biomol Eng 22(4):105–12

    Article  CAS  Google Scholar 

  • Patrick WM, Firth AE, Blackburn JM (2003) User-friendly algorithms for estimating completeness and diversity in randomized protein-encoding libraries. Protein Eng 16(6):451–7

    Article  CAS  Google Scholar 

  • Radestock S, Gohlke H (2011) Protein rigidity and thermophilic adaptation. Proteins 79(4):1089–108

    Article  CAS  Google Scholar 

  • Reetz MT, Carballeira JD (2007) Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes. Nat Protoc 2(4):891–903

    Article  CAS  Google Scholar 

  • Reetz MT, Carballeira JD, Vogel A (2006) Iterative saturation mutagenesis on the basis of B factors as a strategy for increasing protein thermostability. Angew Chem Int Ed Engl 45(46):7745–51

    Article  CAS  Google Scholar 

  • Reetz MT, Prasad S, Carballeira JD, Gumulya Y, Bocola M (2010) Iterative saturation mutagenesis accelerates laboratory evolution of enzyme stereoselectivity: rigorous comparison with traditional methods. J Am Chem Soc 132(26):9144–52

    Article  CAS  Google Scholar 

  • Sambrook J, Russell DW (2001) Molecular cloning: A laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor

    Google Scholar 

  • Schymkowitz J, Borg J, Stricher F, Nys R, Rousseau F, Serrano L (2005) The FoldX web server: an online force field. Nucleic Acids Res 33 (Web Server issue):W382-8

  • Stephens DE, Singh S, Permaul K (2009) Error-prone PCR of a fungal xylanase for improvement of its alkaline and thermal stability. FEMS Microbiol Lett 293(1):42–7

    Article  CAS  Google Scholar 

  • Talakad JC, Wilderman PR, Davydov DR, Kumar S, Halpert JR (2010) Rational engineering of cytochromes P450 2B6 and 2B11 for enhanced stability: insights into structural importance of residue 334. Arch Biochem Biophys 494(2):151–158

    Article  CAS  Google Scholar 

  • Tian J, Wu N, Guo X, Guo J, Zhang J, Fan Y (2007) Predicting the phenotypic effects of non-synonymous single nucleotide polymorphisms based on support vector machines. BMC Bioinforma 8:450

    Article  Google Scholar 

  • Tian J, Wang P, Gao S, Chu X, Wu N, Fan Y (2010a) Enhanced thermostability of methyl parathion hydrolase from Ochrobactrum sp. M231 by rationally engineering a glycine to proline mutation. FEBS J 277:4901–4908

    Article  CAS  Google Scholar 

  • Tian J, Wu N, Chu X, Fan Y (2010b) Predicting changes in protein thermostability brought about by single- or multi-site mutations. BMC Bioinforma 11:370

    Article  Google Scholar 

  • Vieille C, Zeikus GJ (2001) Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol Mol Biol Rev 65(1):1–43

    Article  CAS  Google Scholar 

  • Xiao W, Chu X, Tian J, Guo J, Wu N (2008) Cloning of a Methyl Parathion Hydrolase Gene from Ochrobactrum sp. Journal of Agricultural Science and Technology (In Chinese)(S1):99-102

  • Yang C, Liu N, Guo X, Qiao C (2006) Cloning of mpd gene from a chlorpyrifos-degrading bacterium and use of this strain in bioremediation of contaminated soil. FEMS Microbiol Lett 265(1):118–25

    Article  CAS  Google Scholar 

  • Yang C, Freudl R, Qiao C, Mulchandani A (2010) Cotranslocation of methyl parathion hydrolase to the periplasm and of organophosphorus hydrolase to the cell surface of Escherichia coli by the Tat pathway and ice nucleation protein display system. Appl Environ Microbiol 76(2):434–40

    Article  CAS  Google Scholar 

  • Yin S, Ding F, Dokholyan NV (2007) Eris: an automated estimator of protein stability. Nat Methods 4(6):466–7

    Article  CAS  Google Scholar 

  • Yu H, Yan X, Shen W, Hong Q, Zhang J, Shen Y, Li S (2009) Expression of methyl parathion hydrolase in Pichia pastoris. Curr Microbiol 59(6):573–8

    Article  CAS  Google Scholar 

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Acknowledgments

We greatly thank Prof. Runsheng Chen for his assistance with bioinformatics. This work was supported by grants from the National Natural Science Foundation of China (grant nos. 30900839 and 31100049).

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Authors and Affiliations

  1. Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, 12 Zhongguancun South Street, Beijing, 100081, China

    Jian Tian, Ping Wang, Lu Huang, Xiaoyu Chu, Ningfeng Wu & Yunliu Fan

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  1. Jian Tian
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  2. Ping Wang
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  3. Lu Huang
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  4. Xiaoyu Chu
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  5. Ningfeng Wu
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  6. Yunliu Fan
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Correspondence to Ningfeng Wu.

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Tian, J., Wang, P., Huang, L. et al. Improving the thermostability of methyl parathion hydrolase from Ochrobactrum sp. M231 using a computationally aided method. Appl Microbiol Biotechnol 97, 2997–3006 (2013). https://doi.org/10.1007/s00253-012-4411-7

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  • DOI: https://doi.org/10.1007/s00253-012-4411-7

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