OptZyme: Computational Enzyme Redesign Using Transition State Analogues

OptZyme is a new computational procedure for designing improved enzymatic activity (i.e., kcat or kcat/KM) with a novel substrate. The key concept is to use transition state analogue compounds, which are known for many reactions, as proxies for the typically unknown transition state structures. Mutations that minimize the interaction energy of the enzyme with its transition state analogue, rather than with its substrate, are identified that lower the transition state formation energy barrier. Using Escherichia coli β-glucuronidase as a benchmark system, we confirm that KM correlates (R2 = 0.960) with the computed interaction energy between the enzyme and the para-nitrophenyl- β, D-glucuronide substrate, kcat/KM correlates (R2 = 0.864) with the interaction energy of the transition state analogue, 1,5-glucarolactone, and kcat correlates (R2 = 0.854) with a weighted combination of interaction energies with the substrate and transition state analogue. OptZyme is subsequently used to identify mutants with improved KM, kcat, and kcat/KM for a new substrate, para-nitrophenyl- β, D-galactoside. Differences between the three libraries reveal structural differences that underpin improving KM, kcat, or kcat/KM. Mutants predicted to enhance the activity for para-nitrophenyl- β, D-galactoside directly or indirectly create hydrogen bonds with the altered sugar ring conformation or its substituents, namely H162S, L361G, W549R, and N550S.OptZyme是一种全新的计算方法，可用于针对新型底物设计提升酶活性（即催化常数kcat或催化效率kcat/KM）的突变体。其核心思路是利用诸多反应中已被探明的过渡态类似物（transition state analogue），作为通常未知的过渡态结构的替代模型。本研究通过筛选能最小化酶与其过渡态类似物之间相互作用能的氨基酸突变（而非酶与底物的相互作用），从而降低过渡态形成的能垒。本研究以大肠杆菌（Escherichia coli）β-葡萄糖醛酸苷酶（β-glucuronidase）作为基准测试系统，验证得到以下结论：米氏常数KM与酶和底物对硝基苯基-β-D-葡萄糖醛酸苷（para-nitrophenyl-β,D-glucuronide）之间的计算相互作用能呈相关性，决定系数R²=0.960；催化效率kcat/KM与过渡态类似物1,5-葡萄糖酸内酯（1,5-glucarolactone）的相互作用能呈相关性，R²=0.864；而催化常数kcat则与底物和过渡态类似物的相互作用能加权组合呈相关性，R²=0.854。随后，本研究借助OptZyme筛选出可针对新型底物对硝基苯基-β-D-半乳糖苷（para-nitrophenyl-β,D-galactoside）提升KM、kcat及kcat/KM的突变体。三类突变文库间的差异揭示了分别提升KM、kcat或kcat/KM的结构性基础。经预测可增强对硝基苯基-β-D-半乳糖苷酶活性的突变体（包括H162S、L361G、W549R及N550S），可直接或间接与改变后的糖环构象及其取代基形成氢键。