title: Effects of point mutations on the thermostability of B. subtilis lipase: investigating nonadditivity.
abstract: Molecular level understanding of mutational effects on stability and activity of enzymes is challenging particularly when several point mutations are incorporated during the directed evolution experiments. In our earlier study, we have suggested the lack of consistency in the effect of point mutations incorporated during the initial generations of directed evolution experiments, towards conformational stabilization of B. subtilis lipase mutants of later generations. Here, we report that the cumulative point mutations incorporated in mutants 2M (with two point mutations) to 6M (with six point mutations) possibly do not retain their original stabilizing nature in the most thermostable 12M mutant (with 12 point mutations). We have carried out MD simulations using structures incorporating reversal of different sets of point mutations to assess their effect on the conformational stability and activity of 12M. Our analysis has revealed that reversal of certain point mutations in 12M had little effect on its conformational stability, suggesting that these mutations were probably inconsequential towards the thermostability of the 12M mutant. Interestingly these mutations involved evolutionarily conserved residues. On the other hand, some of the other point mutations incorporated in nonconserved regions, appeared to contribute significantly towards the conformational stability and/or activity of 12M. Based on the analysis of dynamics of in silico mutants generated using the consensus sequence, we identified experimentally verifiable residue positions to further increase the conformational stability and activity of the 12M mutant.
authors.affiliation: Center for Computational Natural Sciences and Bioinformatics (CCNSB), International Institute of Information Technology Hyderabad (IIIT-H), Gachibowli, Hyderabad, 500032, India.
authors.forename: Bipin
authors.initials: B
authors.lastname: Singh
authors.affiliation: Center for Computational Natural Sciences and Bioinformatics (CCNSB), International Institute of Information Technology Hyderabad (IIIT-H), Gachibowli, Hyderabad, 500032, India. gopal@atc.tcs.com.|TCS Innovation Labs-Hyderabad (Life Sciences Division), Tata Consultancy Services Limited, Madhapur, Hyderabad, 500081, India. gopal@atc.tcs.com.
authors.forename: Gopalakrishnan
authors.initials: G
authors.lastname: Bulusu
authors.affiliation: Center for Computational Natural Sciences and Bioinformatics (CCNSB), International Institute of Information Technology Hyderabad (IIIT-H), Gachibowli, Hyderabad, 500032, India.
authors.forename: Abhijit
authors.initials: A
authors.lastname: Mitra
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D055808:Drug Discovery; D004795:Enzyme Stability; D006358:Hot Temperature; D008049:Lipase / Q000737:chemistry* / Q000235:genetics; D056004:Molecular Dynamics Simulation; D017354:Point Mutation; D011487:Protein Conformation; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D008049:Lipase
keywords: Conserved/non-conserved residues; Directed evolution; Free energy landscape; In silico mutagenesis; Molecular dynamics simulations; Protein engineering