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Compensatory Stabilizing Role of Surface Mutations During the Directed Evolution of Dienelactone Hydrolase for Enhanced Activity

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Abstract

Directed evolution is a common tool employed to generate enzymes suitable for industrial use. High thermal stability is often advantageous or even a requirement for biocatalysts, as such the evolution of protein stability is of practical as well as academic interest. Even when evolving enzymes for new or improved catalytic functions, stability is an important factor since it can limit the accumulation rate and number of desired active site mutations. Dienelactone hydrolase, a small monomeric protein, has been previously evolved via a three-stage process to possess enhanced activity and specificity toward non-physiological substrates. In addition to seven active site mutations there were three surface mutations that were thought to increase the stability of the enzyme and compensate for the destabilizing active site mutations. Here, the individual influence of the three surface mutations—Q110L, Y137C and N154D—on the thermal and chemical stability of DLH has been assessed. While the Q110L and N154D mutations improved the thermal stability, the influence of the Y137C mutation was more complex. Individually it was destabilizing both thermally and chemically, but when in the presence of the Q110L and N154D mutations its effect was neutralized in relation to thermal but not chemical stability. In the context of a directed evolution experiment, these compensatory surface mutations play important roles. However, our results show that detrimental mutations can arise, thus the simultaneous monitoring of stability changes while evolving enzymes for enhanced catalytic properties can be beneficial.

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Abbreviations

DLH:

Dienelactone hydrolase

PCR:

Polymerase chain reaction

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

Tm :

Melting temperature

References

  1. Polizzi KM, Bommarius AS, Broering JM, Chaparro-Riggers JF (2007) Stability of biocatalysts. Curr Opin Chem Biol 11:220–225

    Article  CAS  Google Scholar 

  2. Bornscheuer UT, Huisman GW, Kazlauskas RJ, Lutz S, Moore JC, Robins K (2012) Engineering the third wave of biocatalysis. Nature 485:185–194

    Article  CAS  Google Scholar 

  3. Turner NJ (2009) Directed evolution drives the next generation of biocatalysts. Nat Chem Biol 5:567–573

    Article  CAS  Google Scholar 

  4. Reetz MT (2011) Laboratory evolution of stereoselective enzymes: a prolific source of catalysts for asymmetric reactions. Angew Chem Int Ed 50:138–174

    Article  CAS  Google Scholar 

  5. Wang M, Si T, Zhao H (2012) Biocatalyst development by directed evolution. Bioresour Technol 115:117–125

    Article  CAS  Google Scholar 

  6. Tracewell CA, Arnold FH (2009) Directed enzyme evolution: climbing fitness peaks one amino acid at a time. Curr Opin Chem Biol 13:3–9

    Article  CAS  Google Scholar 

  7. Bloom JD, Meyer MM, Meinhold P, Otey CR, MacMillan D, Arnold FH (2005) Evolving strategies for enzyme engineering. Curr Opin Struct Biol 15:447–452

    Article  CAS  Google Scholar 

  8. Bloom JD, Labthavikul ST, Otey CR, Arnold FH (2006) Protein stability promotes evolvability. Proc Natl Acad Sci USA 103:5869–5874

    Article  CAS  Google Scholar 

  9. Tokuriki N, Tawfik DS (2009) Stability effects of mutations and protein evolvability. Curr Opin Struct Biol 19:596–604

    Article  CAS  Google Scholar 

  10. Pathak D, Ollis D (1990) Refined structure of dienelactone hydrolase at 1.8 Å. J Mol Biol 214:497–525

    Article  CAS  Google Scholar 

  11. Porter JL, Carr PD, Collyer CA, Ollis DL (2014) Crystallization of dienelactone hydrolase in two space groups: structural changes caused by crystal packing. Acta Crystallogr Sect F: Struct Biol Cryst Commun 70:884–889

    Article  CAS  Google Scholar 

  12. Porter JL, Boon PLS, Murray TP, Huber T, Collyer CA, Ollis DL (2014) Directed evolution of new and improved enzyme functions using an evolutionary intermediate and multidirectional search. ACS Chem Biol. doi:10.1021/cb500809f

    Google Scholar 

  13. Liu J-W, Boucher Y, Stokes HW, Ollis DL (2006) Improving protein solubility: the use of the Escherichia coli dihydrofolate reductase gene as a fusion reporter. Protein Expr Purif 47:258–263

    Article  CAS  Google Scholar 

  14. Gasteiger E, Hoogland C, Gattiker A, Se D, Wilkins M, Appel R, Bairoch A (2005) Protein identification and analysis tools on the ExPASy server. In: Walker J (ed) The proteomics protocols handbook. Humana Press, New York, pp 571–607

    Chapter  Google Scholar 

  15. Krissinel E, Henrick K (2004) Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr Sect D: Biol Crystallogr 60:2256–2268

    Article  CAS  Google Scholar 

  16. Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, Keegan RM, Krissinel EB, Leslie AGW, McCoy A, McNicholas SJ, Murshudov GN, Pannu NS, Potterton EA, Powell HR, Read RJ, Vagin A, Wilson KS (2011) Overview of the CCP4 suite and current developments. Acta Crystallogr Sect D: Biol Crystallogr 67:235–242

    Article  CAS  Google Scholar 

  17. Johnson KA, Goody RS (2011) The original Michaelis constant: translation of the 1913 Michaelis–Menten paper. Biochemistry 50:8264–8269

    Article  CAS  Google Scholar 

  18. Niesen FH, Berglund H, Vedadi M (2007) The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat Protoc 2:2212–2221

    Article  CAS  Google Scholar 

  19. Levisson M, Oost J, Kengen SWM (2009) Carboxylic ester hydrolases from hyperthermophiles. Extremophiles 13:567–581

    Article  CAS  Google Scholar 

  20. Unsworth LD, van der Oost J, Koutsopoulos S (2007) Hyperthermophilic enzymes—stability, activity and implementation strategies for high temperature applications. FEBS J 274:4044–4056

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  22. Richardson JS (1981) The anatomy and taxonomy of protein structure. Adv Protein Chem 34:167–339

    Article  CAS  Google Scholar 

  23. Barlow DJ, Thornton JM (1988) Helix geometry in proteins. J Mol Biol 201:601–619

    Article  CAS  Google Scholar 

  24. Stevenson BJ, Liu J-W, Ollis DL (2008) Directed evolution of yeast pyruvate decarboxylase 1 for attenuated regulation and increased stability. Biochemistry 47:3013–3025

    Article  CAS  Google Scholar 

  25. Rossky PJ (2008) Protein denaturation by urea: slash and bond. Proc Natl Acad Sci USA 105:16825–16826

    Article  CAS  Google Scholar 

  26. Bennion BJ, Daggett V (2003) The molecular basis for the chemical denaturation of proteins by urea. Proc Natl Acad Sci USA 100:5142–5147

    Article  CAS  Google Scholar 

  27. Hua L, Zhou R, Thirumalai D, Berne BJ (2008) Urea denaturation by stronger dispersion interactions with proteins than water implies a 2-stage unfolding. Proc Natl Acad Sci USA 105:16928–16933

    Article  CAS  Google Scholar 

  28. Canchi DR, Paschek D, Garcia AE (2010) Equilibrium study of protein denaturation by urea. J Am Chem Soc 132:2338–2344

    Article  CAS  Google Scholar 

  29. Moeser B, Horinek D (2014) Unified description of urea denaturation: backbone and side chains contribute equally in the transfer model. J Phys Chem B 118:107–114

    Article  CAS  Google Scholar 

  30. Ramprakash J, Doseeva V, Galkin A, Krajewski W, Muthukumar L, Pullalarevu S, Demirkan E, Herzberg O, Moult J, Schwarz FP (2008) Comparison of the chemical and thermal denaturation of proteins by a two-state transition model. Anal Biochem 374:221–230

    Article  CAS  Google Scholar 

  31. Cowan DA (1997) Thermophilic proteins: stability and function in aqueous and organic solvents. Comp Biochem Physiol Part A: Mol Integr Physiol 118A:429–438

    Article  CAS  Google Scholar 

  32. Doukyu N, Ogino H (2010) Organic solvent-tolerant enzymes. Biochem Eng J 48:270–282

    Article  CAS  Google Scholar 

Download references

Acknowledgments

JLP is supported by an Australian Postgraduate Award from the Australian government and funding from the Research School of Chemistry, ANU. We thank the staff at the ACRF-BRF for performing the DNA sequencing analysis. We also thank Anithahini Jeyasingham from the Mass Spectrometry Department in the Research School of Chemistry at the ANU for conducting the mass spectrometry.

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Authors and Affiliations

  1. Research School of Chemistry, Australian National University, Canberra, ACT, 2601, Australia

    Joanne L. Porter & David L. Ollis

  2. School of Molecular Bioscience, University of Sydney, Sydney, NSW, 2006, Australia

    Charles A. Collyer

Authors
  1. Joanne L. Porter
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  2. Charles A. Collyer
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  3. David L. Ollis
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Correspondence to Joanne L. Porter.

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Porter, J.L., Collyer, C.A. & Ollis, D.L. Compensatory Stabilizing Role of Surface Mutations During the Directed Evolution of Dienelactone Hydrolase for Enhanced Activity. Protein J 34, 82–89 (2015). https://doi.org/10.1007/s10930-015-9600-7

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  • DOI: https://doi.org/10.1007/s10930-015-9600-7

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