ИСТИНА

Recently developed methodology for rational design of biocatalysts based on bioinformatic analysis of enzyme superfamilies and molecular modeling/computer screening of potential mutants was applied to improve stability and catalytic performance of penicillin acylase from Escherichia coli. Subfamily-specific positions responsible for variation of functional properties among penicillin acylase family enzymes were identified using bioinformatic analysis [1-3] (computer program Zebra at http://biokinet.belozersky.msu.ru/zebra) and were considered as hotspots for rational design of enzyme mutants. Molecular dynamics was exploited to simulate pH-dependent inactivation and compare stability of the wild type penicillin acylase and its variants. Subfamily-specific position bD484 was identified as a key element of the buried side chain interaction network, collapse of which at alkaline pH disturbs a native protein conformation. The single stabilizing substitution bD484N has been proposed by bioinformatic analysis of homologous Ntn-hydrolases with different pH stability. The corresponding bD484N mutant was much more stable in alkaline medium (9-fold improvement at pH 10.0) [4], however even more important was stabilization to the earlier observed enzyme inactivation at high substrate concentrations [5]. Designed mutation allowed to improve catalytic performance of penicillin acylase at biocatalytic acylation of amino acids and peptide synthesis in aqueous medium and had led to 5-fold increased yield of the preparative D-phenylglycine-derived peptide synthesis from equimolar substrate mixtures compared to the wild type enzyme [6]. The above mentioned strategy for rational design of biocatalysts can be further explored to engineer enzyme variants with improved functional properties. This work was supported by the Russian Foundation for Basic Research, grant 14-08-00987. References 1 Suplatov D., Shalaeva D., Kirilin E., Arzhanik V., Švedas V. Bioinformatic analysis of protein families for identification of variable amino acid residues responsible for functional diversity. Journal of Biomolecular Structure and Dynamics. 2014, 32(1), 75-87. 2 Suplatov D., Kirilin E., Takhaveev V., Švedas V. Zebra: a web server for bioinformatic analysis of diverse protein families. Journal of Biomolecular Structure and Dynamics, 2014, 32(11), 1752-1758. 3 Suplatov D.A., Voevodin V.V., Švedas V.K., Robust enzyme design: Bioinformatic tools for improved protein stability, Biotechnology J., 2015, 10(3), 344-355. 4 Suplatov D., Panin N., Kirilin E., Shcherbakova T., Kudryavtsev P., Švedas V., Computational design of a pH stable enzyme: understanding molecular mechanism of penicillin acylase’s adaptation to alkaline conditions, PLoS ONE 2014, 9(6): e100643. 5 Shcherbakova T.A., Korennykh A.V., van Langen L.M., Sheldon R.A., Švedas V.K. Use of high acyl donor concentrations leads to penicillin acylase inactivation in the course of peptide synthesis, J. Mol. Cat. B: Enzym. 2004, 31, 63-65. 6 Shcherbakova T.A., Panin N.V., Suplatov D.A., Shapovalova I.V., Svedas V.K., The βD484N mutant of penicillin acylase from Escherichia coli is more resistant to inactivation by substrates and can effectively perform peptide synthesis in aqueous medium, J. Mol. Cat. B: Enzym., 2015, 112, 66–68.