College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30 Puzhu South Road, Nanjing 211816, China; College of Biological and Pharmaceutical Engineering, Research Center of Natural Medicine and Traditional Chinese Medicine, West Anhui University, Lu'an 237012, China.
School of Pharmaceutical Sciences, Nanjing Tech University, No. 30 Puzhu South Road, Nanjing 211816, China.
Food Chem. 2018 Jul 1;253:30-36. doi: 10.1016/j.foodchem.2018.01.108. Epub 2018 Jan 31.
Metalloprotease PT121, an effective catalyst for Z-aspartame synthesis under the substrate (Z-Asp:l-Phe-OMe) molar ratio of 1:5, was obtained previously. Herein, a computational strategy combining molecular dynamics simulation of the enzyme-substrate complex with binding free energy (ΔG) calculations was established to guide the further engineering of PT121. One His224 residue proximal to the PT121 active site was selected on the basis of the difference in ΔG decomposition of PT121 toward l-Phe-NH and l-Phe-OMe. Site-saturation mutagenesis of His224 resulted in the mutants H224D, H224N, and H224S, which showed great improvement in Z-aspartame synthesis under an economical substrate molar ratio approaching 1:1. Furthermore, the kinetic constants of PT121 and its mutants revealed that the affinity of mutants toward the l-Phe-OMe was significantly higher than that of PT121. Molecular dynamic simulation revealed that the enhanced synthetic activity may be attributed to the mutation stabilizing the transient state of the enzyme-l-Phe-OMe complex.
先前获得了一种金属蛋白酶 PT121，它在（Z-Asp：l-Phe-OMe）摩尔比为 1:5 的条件下是合成 Z-天冬甜素的有效催化剂。在此，建立了一种结合酶-底物复合物的分子动力学模拟和结合自由能（ΔG）计算的计算策略，以指导 PT121 的进一步工程改造。根据 PT121 对 l-Phe-NH 和 l-Phe-OMe 的 ΔG 分解的差异，选择了靠近 PT121 活性位点的一个 His224 残基。对 His224 进行定点饱和突变得到了 H224D、H224N 和 H224S 突变体，它们在接近 1:1 的经济底物摩尔比下进行 Z-天冬甜素合成时表现出了巨大的改善。此外，PT121 及其突变体的动力学常数表明，突变体对 l-Phe-OMe 的亲和力明显高于 PT121。分子动力学模拟表明，增强的合成活性可能归因于突变稳定了酶-l-Phe-OMe 复合物的瞬态。
