Departament de Química Inorgànica i Orgànica (Secció de Química Orgànica), Universitat de Barcelona, Barcelona, Spain.
Institut de Quimica Teorica i Computacional (IQTCUB), Universitat de Barcelona, Barcelona, Spain.
Nat Chem Biol. 2017 Aug;13(8):874-881. doi: 10.1038/nchembio.2394. Epub 2017 Jun 12.
Si-like mechanisms, which involve front-face leaving group departure and nucleophile approach, have been observed experimentally and computationally in chemical and enzymatic substitution at α-glycosyl electrophiles. Since Si-like, S1 and S2 substitution pathways can be energetically comparable, engineered switching could be feasible. Here, engineering of Sulfolobus solfataricus β-glycosidase, which originally catalyzed double S2 substitution, changed its mode to Si-like. Destruction of the first S2 nucleophile through E387Y mutation created a β-stereoselective catalyst for glycoside synthesis from activated substrates, despite lacking a nucleophile. The pH profile, kinetic and mutational analyses, mechanism-based inactivators, X-ray structure and subsequent metadynamics simulations together suggest recruitment of substrates by π-sugar interaction and reveal a quantum mechanics-molecular mechanics (QM/MM) free-energy landscape for the substitution reaction that is similar to those of natural, Si-like glycosyltransferases. This observation of a front-face mechanism in a β-glycosyltransfer enzyme highlights that Si-like pathways may be engineered in catalysts with suitable environments and suggests that 'β-Si' mechanisms may be feasible for natural glycosyltransfer enzymes.
类似硅的机制，涉及前侧离去基团的离去和亲核试剂的接近，在α-糖苷基亲电试剂的化学和酶取代中已经通过实验和计算得到了观察。由于类似硅的、S1 和 S2 取代途径在能量上可能是可比的，因此工程切换是可行的。在这里，对最初催化双 S2 取代的 Sulfolobus solfataricus β-糖苷酶进行了工程改造，将其模式改变为类似硅的模式。通过 E387Y 突变破坏第一个 S2 亲核试剂，创建了一个用于从激活底物进行糖苷合成的β-立体选择性催化剂，尽管缺乏亲核试剂。pH 曲线、动力学和突变分析、基于机制的失活剂、X 射线结构和随后的元动力学模拟共同表明通过π-糖相互作用募集底物，并揭示了取代反应的量子力学-分子力学（QM/MM）自由能景观，类似于天然的、类似硅的糖基转移酶。在β-糖苷基转移酶中观察到的前侧机制突出表明，类似硅的途径可以在具有合适环境的催化剂中进行工程改造，并表明“β-Si”机制可能适用于天然糖基转移酶。
