Engineering DszC Mutants from Transition State Macrodipole Considerations and Evolutionary Sequence Analysis

We describe an approach to identify enzyme mutants with increased turnover using the enzyme DszC as a case study. Our approach is based on recalculating the barriers of alanine mutants through single-point energy calculations at the hybrid QM/MM level in the wild-type reactant and transition state geometries. We analyze the difference in the electron density between the reactant and transition state to identify sites/residues where electrostatic interactions stabilize the transition state over the reactants. We also assess the insertion of a unit probe charge to identify positions in which the introduction of charged residues lowers the barrier.本研究以酶DszC为研究案例，提出了一种可识别催化周转数提升的酶突变体的方法。该方法基于在野生型反应物与过渡态几何结构下，采用杂化量子力学/分子力学（QM/MM）理论水平的单点能计算，对丙氨酸突变体的活化能垒进行重新计算。我们通过分析反应物与过渡态间的电子密度差异，识别出那些可通过静电相互作用使过渡态相较于反应物更稳定的位点/残基。此外，我们还通过引入单位探针电荷的方式开展评估，以筛选出引入带电残基可降低活化能垒的位点。