Thermostabilizing mechanisms of canonical single amino acid substitutions at a GH1 β-glucosidase probed by multiple MD and computational approaches

作     者：Oliveira Rocha, Rafael Eduardo Batista Mariano, Diego Cesar Almeida, Tiago Silva CorreaCosta, Leon Sulfierry Camargo Fischer, Pedro Henrique Santos, Lucianna Helene Caffarena, Ernesto Raul da Silveira, Carlos Henrique Lamp, Leonida M. Fernandez-Quintero, Monica Lisa Liedl, Klaus Roman De Melo-Minardi, Raquel Cardoso Franca de Lima, Leonardo Henrique 

作者机构：Univ Fed Sao Joao del Rei Dept Phys & Biol Sci Lab Mol Modelling & Bioinformat LAMMB Campus Sete Lagoas BR-35701970 Sete Lagoas MG Brazil Univ Fed Minas Gerais Dept Comp Sci Lab Bioinformat & Syst LBS Belo Horizonte MG Brazil Univ Fed Minas Gerais Dept Biochem & Immunol Lab Mol Modeling & Drug Design Belo Horizonte MG Brazil Lab Nacl Comp Cient LNCC Computat Modeling Coordinat COMOD Petropolis RJ Brazil Fiocruz MS Sci Comp Program Rio De Janeiro Brazil Univ Fed Itajuba Technol Sci Inst Itabira MG Brazil Univ Innsbruck Inst Gen Inorgan & Theoret Chem Innsbruck Austria Univ Innsbruck Ctr Chem & Biomed Innsbruck CCB Innsbruck Austria 

出 版 物：《PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS》 (蛋白质：结构、性能和生物信息学)

年 卷 期：2023年第91卷第2期

页      面：218-236页

核心收录：

学科分类：0710[理学-生物学] 071010[理学-生物化学与分子生物学] 07[理学] 

基　　金：Conselho Nacional de Desenvolvimento Cientifico e Tecnologico Fundacao de Amparo a Pesquisa do Estado de Minas Gerais Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior 

主　　题：accelerated biofuel beta-glucosidase enzymes conformational entropy evolutive residue coupling grid inhomogeneous solvation theory molecular dynamics metadynamics molecular dynamics Poisson-Boltzmann surface analysis thermostabilizing mutations 

摘      要：beta-glucosidases play a pivotal role in second-generation biofuel (2G-biofuel) production. For this application, thermostable enzymes are essential due to the denaturing conditions on the bioreactors. Random amino acid substitutions have originated new thermostable beta-glucosidases, but without a clear understanding of their molecular mechanisms. Here, we probe by different molecular dynamics simulation approaches with distinct force fields and submitting the results to various computational analyses, the molecular bases of the thermostabilization of the Paenibacillus polymyxa GH1 beta-glucosidase by two-point mutations E96K (TR1) and M4161 (TR2). Equilibrium molecular dynamic simulations (eMD) at different temperatures, principal component analysis (PCA), virtual docking, metadynamics (MetaDy), accelerated molecular dynamics (aMD), Poisson-Boltzmann surface analysis, grid inhomogeneous solvation theory and colony method estimation of conformational entropy allow to converge to the idea that the stabilization carried by both substitutions depend on different contributions of three classic mechanisms: (i) electrostatic surface stabilization;(ii) efficient isolation of the hydrophobic core from the solvent, with energetic advantages at the solvation cap;(iii) higher distribution of the protein dynamics at the mobile active site loops than at the protein core, with functional and entropic advantages. Mechanisms i and ii predominate for TR1, while in TR2, mechanism iii is dominant. Loop A integrity and loops A, C, D, and E dynamics play critical roles in such mechanisms. Comparison of the dynamic and topological changes observed between the thermostable mutants and the wildtype protein with amino acid coevolutive networks and thermostabilizing hotspots from the literature allow inferring that the mechanisms here recovered can be related to the thermostability obtained by different substitutions along the whole family GH1. We hope the results and insights discussed her