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Rational design of penicillin acylase from Escherichia coli based on bioinformatic analysis of Ntn-hydrolase superfamily of enzymes and molecular modeling of potential mutants was carried out to improve stability and catalytic performance in alkaline medium. Recently developed method of bioinformatic analysis [1-3] and corresponding computer program Zebra [http://biokinet.belozersky.msu.ru/zebra] were used to identify subfamily-specific positions that are supposed to be responsible for discrimination of functional properties of Ntn-hydrolases and were considered as hotspots for mutation. Molecular dynamics was used to evaluate stability and simulate pH-dependent inactivation of the wild type enzyme and its mutants. Molecular modeling was used to compare conformational spaces of penicillin acylase from Escherichia coli in aqueous media at neutral and alkaline conditions and reveal the subfamily-specific ionazible residues with buried side-chains which are related to unfolding of the native protein conformation. Mechanism of pH-induced inactivation was studied and subfamily-specific position βD484 was identified as a key element of the crucial buried interaction network, which collapse at alkaline pH disturbs the native protein conformation. The βD484 residue was chosen as a hotspot for mutation to engineer more stable enzyme variant in alkaline medium: single stabilizing substitution βD484N has been proposed by bioinformatic analysis of homologous Ntn-hydrolases with different pH stability; corresponding mutant was expressed, purified and characterized experimentally. The βD484N mutation substantially stabilized penicillin acylase from Escherichia coli in alkaline medium (10-fold improvement at pH 10.0) however even more important was enzyme stabilization to inactivation at high substrate concentration [4]. Observed stabilization effects allowed to improve catalytic performance of penicillin acylase at enzymatic peptide synthesis in aqueous medium and leaded to 5-fold increased yield of preparative D-phenylglycine-derived peptide synthesis from equimolar substrate mixtures compared to the wild type enzyme. We suggest that bioinformatic analysis of subfamily-specific positions should be further explored to study the mechanisms of protein stability and to design stable variants as a prerequisite to engineering catalytic activity, substrate specificity, stereoselectivity, etc. of novel enzymes 1. Suplatov D., Shalaeva D., Kirilin E., Arzhanik V., Švedas V. (2013) Bioinformatic analysis of protein families for identification of variable amino acid residues responsible for functional diversity. J Biomol Struct Dyn,, DOI:10.1080/07391102.2012.750249 2. Suplatov D., Besenmatter W., Švedas V., Svendsen A. (2012) Bioinformatic analysis of α/β-hydrolase fold enzymes reveals subfamily-specific positions responsible for discrimination of amidase and lipase activities. Protein Eng Des Sel., 25(11), 689-697 3. Suplatov D., Arzhanik V., Švedas V. (2011) Comparative Bioinformatic Analysis of Active Site Structures in Evolutionarily Remote Homologues of α,β-Hydrolase Superfamily Enzymes, Acta Naturae, 3(1), 93-98. 4. Shcherbakova T.A., Korennykh A.V., van Langen L.M., Sheldon R.A., Švedas V.K. (2004) Use of high acyl donor concentrations leads to penicillin acylase inactivation in the course of peptide synthesis, J Mol Cat B: Enzym., 31, 63-65.