title: Computational design of a pH stable enzyme: understanding molecular mechanism of penicillin acylase's adaptation to alkaline conditions.
abstract: Protein stability provides advantageous development of novel properties and can be crucial in affording tolerance to mutations that introduce functionally preferential phenotypes. Consequently, understanding the determining factors for protein stability is important for the study of structure-function relationship and design of novel protein functions. Thermal stability has been extensively studied in connection with practical application of biocatalysts. However, little work has been done to explore the mechanism of pH-dependent inactivation. In this study, bioinformatic analysis of the Ntn-hydrolase superfamily was performed to identify functionally important subfamily-specific positions in protein structures. Furthermore, the involvement of these positions in pH-induced inactivation was studied. The conformational mobility of penicillin acylase in Escherichia coli was analyzed through molecular modeling in neutral and alkaline conditions. Two functionally important subfamily-specific residues, Gluβ482 and Aspβ484, were found. Ionization of these residues at alkaline pH promoted the collapse of a buried network of stabilizing interactions that consequently disrupted the functional protein conformation. The subfamily-specific position Aspβ484 was selected as a hotspot for mutation to engineer enzyme variant tolerant to alkaline medium. The corresponding Dβ484N mutant was produced and showed 9-fold increase in stability at alkaline conditions. Bioinformatic analysis of subfamily-specific positions can be further explored to study mechanisms of protein inactivation and to design more stable variants for the engineering of homologous Ntn-hydrolases with improved catalytic properties.
authors: Lomonosov Moscow State University, Belozersky Institute of Physicochemical Biology and Faculty of Bioengineering and Bioinformatics, Moscow, Russia.
authors: Dmitry
authors: D
authors: Suplatov
authors: Lomonosov Moscow State University, Belozersky Institute of Physicochemical Biology and Faculty of Bioengineering and Bioinformatics, Moscow, Russia.
authors: Nikolay
authors: N
authors: Panin
authors: Lomonosov Moscow State University, Belozersky Institute of Physicochemical Biology and Faculty of Bioengineering and Bioinformatics, Moscow, Russia.
authors: Evgeny
authors: E
authors: Kirilin
authors: Lomonosov Moscow State University, Belozersky Institute of Physicochemical Biology and Faculty of Bioengineering and Bioinformatics, Moscow, Russia.
authors: Tatyana
authors: T
authors: Shcherbakova
authors: Lomonosov Moscow State University, Belozersky Institute of Physicochemical Biology and Faculty of Bioengineering and Bioinformatics, Moscow, Russia.
authors: Pavel
authors: P
authors: Kudryavtsev
authors: Lomonosov Moscow State University, Belozersky Institute of Physicochemical Biology and Faculty of Bioengineering and Bioinformatics, Moscow, Russia.
authors: Vytas
authors: V
authors: Svedas
mesh_terms: D000222:Adaptation, Physiological; D019943:Amino Acid Substitution; D004789:Enzyme Activation; D004795:Enzyme Stability; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009154:Mutation; D010399:Penicillin Amidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D010399:Penicillin Amidase