Computational Design of Nitrile Hydratase from Pseudonocardia thermophila JCM3095 for Improved Thermostability

Abstract

High thermostability and catalytic activity are key properties for nitrile hydratase (NHase, EC 4.2.1.84) as a well-industrialized catalyst. In this study, rational design was applied to tailor the thermostability of NHase from Pseudonocardia thermophila JCM3095 (PtNHase) by combining FireProt server prediction and molecular dynamics (MD) simulation. Site-directed mutagenesis of non-catalytic residues provided by the rational design was subsequentially performed. The positive multiple-point mutant, namely, M10 (αI5P/αT18Y/αQ31L/αD92H/βA20P/βP38L/βF118W/βS130Y/βC189N/βC218V), was obtained and further analyzed. The Melting temperature (Tm) of the M10 mutant showed an increase by 3.2 °C and a substantial increase in residual activity of the enzyme at elevated temperatures was also observed. Moreover, the M10 mutant also showed a 2.1-fold increase in catalytic activity compared with the wild-type PtNHase. Molecular docking and MD simulations demonstrated better substrate affinity and improved thermostability for the mutant.

Development of thermostable sucrose phosphorylase by semi-rational design for efficient biosynthesis of alpha-D-glucosylglycerol

“…Such creation of hydrophobic cores was observed during analysis of mesophilic and thermophilic proteins sequences (Haney et al 1999 ; Saelensminde et al 2009 ). During the process of MD simulations, when residue is mutated into hydrophobic amino acid, the hydrophobic core can be formed, such as in neurotensin receptor (Lee et al 2015 ), enzymes like trehalose synthase (Chen et al 2021 ), and nitrile hydratase (Cheng et al 2020 ; Guo et al 2021 ). The semi-rational design approach established in the present study sheds light on the future engineering of other industrial attractive SPases, such as the one from Bifidobacterium adolescentis.…”

“…The semi-rational design combines the benefits of directed evolution and rational design and is suitable for modifying proteins without high-throughput determination methods and structure–function understanding (Chica et al 2005 ). Many successful examples have reported that changing a few amino acids can significantly improve the protein thermostability by semi-rational design strategies (Cheng et al 2020 ; Roth et al 2017 ).…”

Improving Thermostability and Catalytic Activity of Glycosyltransferase From Panax ginseng by Semi-Rational Design for Rebaudioside D Synthesis

“…Improving thermostability may decrease the enzyme activity because it could change the flexibility of the structure ( Xu et al, 2020 ). But, there are cases in which the thermostability was improved and the enzyme activity was increased by using FireProt ( Cheng et al, 2020 ; Xia et al, 2021 ). Protein analysis tools such as Rate4Site, FoldX, and Rosetta design were assembled to offer a reliable design of stable multiple-point mutants in FireProt ( Musil et al, 2017 ).…”

“…Two distinct methodologies were involved in the semi-rational design: sequence-based enzyme redesign and structure-based enzyme redesign ( Xiong et al, 2021 ). Proteins without a crystal structure or high-throughput determination methods could be redesigned for higher activity and better thermal stability by semi-rational design strategies ( Nakano et al, 2018 ; Cheng et al, 2020 ).…”

NO● Represses the Oxygenation of Arachidonoyl PE by 15LOX/PEBP1: Mechanism and Role in Ferroptosis

“…Conventional full-atomic MD simulations [ 47 , 48 , 49 ] were performed for human 15LOX-2 (PDB id: 4NRE [ 20 ]) and 15LOX-2/PEBP1 complex (PDB ids: 4NRE and 1BEH [ 50 ]), with a bound substrate (SAPE), in the presence of randomly distributed nitric oxide and oxygen molecules with different ratios, such as 1:1 (five of each), 1:3, and 3:1. Multiple MD runs (see Supplementary Table S1 ) of 150 ns with different initial spatial distributions of NO ● and O 2 molecules were performed for each structure (15LOX-2 and its complex with PEBP1) using the NAMD [ 51 ] software with the CHARMM [ 52 ] force field and 2 fs time steps.…”