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.

Robust enzyme design: Bioinformatic tools for improved protein stability

“…Consequently, in most cases this approach limits the size of the set to just a few sequences with verified function and introduces bias. Alternatively, more advanced procedures implement sequence and structure similarity search methods to automatically retrieve homologs from public databases [38,71]. This usually results in a large collection of evolutionary remote proteins which are superimposed by a combination of sequence and structure comparison.…”

“…It was shown that substitutions at the SSPs can lead to catalytic promiscuity of Candida antarctica lipase B [70]. In a different study, mutation of a SSP improved stability of Escherichia coli penicillin acylase in alkaline medium [71]. A multiple structure-guided sequence alignment of a non-redundant set of 230 proteins was used to identify the two most statistically significant subfamily-specific residues which can be in different ionization states at neutral and alkaline environment.…”

Hybrid Computing Clusters To Study Protein Structure, Function And Regulation

“…Таким образом, установлена противотуберкулез-ная активность предложенных соединений. Подходы, аналогичные использованным в этой работе, были ранее применены нами для изучения структуры и функции фермен-тов из других суперсемейств, поиска и характеристики новых центров связывания, а также получения препаратов ферментов с улучшенными свойствами [16][17][18][19][20].…”

Impact of polybasic alcohols on biocompatibility and selectivity of Penicillin G Acylase for kinetically controlled synthesis

“…Protein engineering or directed evolution studies have been used to understand the structure-function relationship and to improve PGA properties such as activity, substrate specificity and stability [7][8][9][10][11][12][13][14][15][16].…”