Understanding the molecular basis of thermostability and activity of B subtilis lipase and its mutants

Abstract

Improving the thermostability of industrial enzymes is an important protein engineering challenge. Molecular level understanding of mutational effects on stability and activity of enzymes is complex particularly when several point mutations are incorporated during the directed evolution experiments, due to non-additivity involving either cooperative (positive) or antagonistic (negative) effects. newlineThe less thermostable mutants (LTMs), 2M to 6M, show WT-like dynamics at all simulation temperatures. However, the two more thermostable mutants 9M and 12M (MTMs) show the required flexibility at appropriate temperature ranges and maintain conformational stability even at high temperature. They show a deep and rugged free-energy landscape, confining them within a near-native conformational space by conserving non-covalent interactions, and thus protecting them from possible aggregation. In contrast, the LTMs having marginally higher thermostabilities than WT show greater probabilities of accessing non-native conformations, which, due to aggregation, have reduced possibilities of reverting to their respective native states under refolding conditions. newlineI 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. My 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. On the other hand, some of the other point mutations incorporated in non-conserved 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, I identified experimentally verifiable residue positions to further increase the conformational stability and activity of the 12M mutant.