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Engineering and introduction of de novo disulphide bridges in organophosphorus hydrolase enzyme for thermostability improvement

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Abstract

The organophosphorus hydrolase (OPH) has been used to degrade organophosphorus chemicals, as one of the most frequently used decontamination methods. Under chemical and thermal denaturing conditions, the enzyme has been shown to unfold. To utilize this enzyme in various applications, the thermal stability is of importance. The engineering of de novo disulphide bridges has been explored as a means to increase the thermal stability of enzymes in the rational method of protein engineering. In this study, Disulphide by Design software, homology modelling and molecular dynamics simulations were used to select appropriate amino acid pairs for the introduction of disulphide bridge to improve protein thermostability. The thermostability of the wild-type and three selected mutant enzymes were evaluated by half-life, ΔG inactivation (ΔGi) and structural studies (fluorescence and far-UV CD analysis). Data analysis showed that half-life of A204C/T234C and T128C/E153C mutants were increased up to 4 and 24 min, respectively; however, for the G74C/A78C mutant, the half-life was decreased up to 9 min. For the T128C/E124C mutant, both thermal stability and Catalytic efficiency (kcat) were also increased. The half-life and ΔGi results were correlated to the obtained information from structural studies by circular dichroism (CD) spectrometry and extrinsic fluorescence experiments; as rigidity increased in A204C/T2234C and T128C/E153C mutants, half-life and ΔGi also increased. For G74C/A78C mutant, these parameters decreased due to its higher flexibility. The results were submitted a strong evidence for the possibility to improve the thermostability of OPH enzyme by introducing a disulphide bridge after bioinformatics design, even though this design would not be always successful.

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Acknowledgements

We thank the manager and colleagues at the Applied Biotechnology Research Centre, Baqiyatallah University of Medical Sciences, Tehran, Iran, for useful discussions and critical reading of the manuscript.

This paper is extracted from Gholamreza Farnoosh’s PhD dissertation of the research project of Applied Biotechnology Research Center of Baqiyatallah University of Medical Sciences.

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Authors and Affiliations

  1. Applied Biotechnology Research Centre, Baqiyatallah University of Medical Sciences, Tehran, Iran

    Gholamreza Farnoosh, Ali Mohammad Latifi & Hossein Aghamollaei

  2. Department of Biochemistry, Faculty of Biologic Science, Tarbiat Modares University, Tehran, Iran

    Khosro Khajeh

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  1. Gholamreza Farnoosh
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  2. Khosro Khajeh
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  3. Ali Mohammad Latifi
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  4. Hossein Aghamollaei
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Correspondence to Ali Mohammad Latifi.

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Corresponding editor: María Eliano Lanio

[Farnoosh G, Khajeh K, Latifi AM and Aghamollaei H 2016 Engineering and introduction of de novo disulphide bridges in organophosphorus hydrolase enzyme for thermostability improvement. J. Biosci.]

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Farnoosh, G., Khajeh, K., Latifi, A.M. et al. Engineering and introduction of de novo disulphide bridges in organophosphorus hydrolase enzyme for thermostability improvement. J Biosci 41, 577–588 (2016). https://doi.org/10.1007/s12038-016-9643-8

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