Simultaneous improvement of the catalytic activity and stability of enzymes is one of the most exciting tasks in biotechnological and pharmaceutical industries. Although many studies have been conducted to figure out their governing factors such as conformational flexibility and strength of electrostatic interactions, accurate prediction of the effects of mutations on the enzyme performance still remains a great challenge. To overcome this issue, a computational methodology has been developed to screen beneficial variants that exhibit enhanced catalytic activity and stability. We divided the enzymes into two regions, tunnel region connecting the enzyme surface to the active site and the surroundings, and evaluated the effect of single-point mutations in each region. At the tunnel region, the amino acids lining the bottleneck which reactants pass through were firstly identified using molecular dynamics (MD) simulation in wild-type (WT) enzyme. Then, the changes in the structure and dynamics of tunnel were computed by substituting the bottleneck residues with other amino acids. The mutations that showed increased frequency of tunnel opening were selected as candidates for experiments because reopening of the tunnel facilitates the access of reactants to the active site, thereby enhancing catalytic activity. Meanwhile, high-fluctuating residues at the surroundings were also examined from the MD trajectories to apply saturation mutagenesis. The relative changes in folding free energy due to point mutations were calculated in order to initially filter out the truly destabilizing mutants. Next, the root mean square fluctuation (RMSF) values were extracted from MD simulations of the mutants selected in the previous step, and subsequently compared with that of WT enzyme to choose the final candidates. It was assumed that reduced RMSF caused by mutations could increase the stability of variants. The performance of this computational methodology has been verified by applying it to several enzymes, including six different dehalogenases. In the best case, a new variant, whose activity and life-time were enhanced by five- and three-times, respectively, was derived by incorporation of two mutations. From this result, validity of the proposed methodology for rational enzyme engineering was demonstrated, and we expect the workflow will contribute to the design of improved variants in terms of the catalytic activity and stability.

<i>In silico</i> enzyme engineering: Computational methodology for improving both the catalytic activity and stability

255th National Meeting (2018)

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In silico enzyme engineering: Computational methodology for improving both the catalytic activity and stability