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Detection of Asp371, Phe375, and Tyr376 Influence on GD-95-10 Lipase Using Alanine Scanning Mutagenesis

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Abstract

GD-95-10 and GD-95-20 lipases are modified GD-95 lipase variants, which lack 10 and 20 C-terminal amino acids, respectively. Previous analysis showed that GD-95-10 lipase has higher activity than GD-95 lipase, while GD-95-20 lipase almost completely loses its activity. Analysis in silico suggested three conservative amino acids at region between 369 and 378 amino acids which can be relevant to the activity of GD-95-10 lipase. These amino acids have direct contacts with residues involved in substrate binding, stabilization of the serine loop or form oxyanion hole. In this work, the role of Asp371, Phe375, and Tyr376 on activity, functionality, and structure of GD-95-10 lipase was analyzed by Ala scanning mutagenesis. We showed that even a single mutation can impact the main structure and activity of Geobacillus lipases. Our experiments provide new knowledge about lipases from thermophilic Geobacillus bacteria and are important for protein engineering and synthetic biology. These enzymes and their engineering can be basis for future biocatalysts applied in production of biofuel or other industrial esters.

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Acknowledgments

This work was supported by the MITA (Agency of Science, Innovation and Technology) program “Development of industrial biotechnology in Lithuania 2011-2013,” project “Innovative tools for cosmetic industry (COSMETIZYM),” Grant No. MITA 31V-18.

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Authors and Affiliations

  1. Department of Microbiology and Biotechnology, Faculty of Natural sciences, Vilnius University, Čiurlionio 21/27, 03101, Vilnius, Lithuania

    Renata Gudiukaitė, Audrius Gegeckas, Mikas Sadauskas & Donaldas Citavicius

Authors
  1. Renata Gudiukaitė
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  2. Audrius Gegeckas
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  3. Mikas Sadauskas
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  4. Donaldas Citavicius
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Corresponding author

Correspondence to Renata Gudiukaitė.

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Table S1

The C-score of GD-95-10 lipase and its mutant 3D models in ProSA-web server (DOC 31 kb)

Table S2

Interactions changes of important amino acids in mutated lipase structures. + - contact detectable; - relationships not detectable (DOC 323 kb)

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Gudiukaitė, R., Gegeckas, A., Sadauskas, M. et al. Detection of Asp371, Phe375, and Tyr376 Influence on GD-95-10 Lipase Using Alanine Scanning Mutagenesis. Appl Biochem Biotechnol 178, 654–669 (2016). https://doi.org/10.1007/s12010-015-1900-z

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  • DOI: https://doi.org/10.1007/s12010-015-1900-z

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