Institut de Química Computacional i Catàlisi and Departament de Química, Universitat de Girona, C/M. Aurèlia Capmany, 69, 17003, Girona, Spain.
Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 E California Blvd., Pasadena, CA 91125, USA.
Angew Chem Int Ed Engl. 2023 Aug 28;62(35):e202303879. doi: 10.1002/anie.202303879. Epub 2023 Jul 20.
We report a computationally driven approach to access enantiodivergent enzymatic carbene N-H insertions catalyzed by P411 enzymes. Computational modeling was employed to rationally guide engineering efforts to control the accessible conformations of a key lactone-carbene (LAC) intermediate in the enzyme active site by installing a new H-bond anchoring point. This H-bonding interaction controls the relative orientation of the reactive carbene intermediate, orienting it for an enantioselective N-nucleophilic attack by the amine substrate. By combining MD simulations and site-saturation mutagenesis and screening targeted to only two key residues, we were able to reverse the stereoselectivity of previously engineered S-selective P411 enzymes. The resulting variant, L5_FL-B3, accepts a broad scope of amine substrates for N-H insertion with excellent yields (up to >99 %), high efficiency (up to 12 300 TTN), and good enantiocontrol (up to 7 : 93 er).
我们报告了一种计算驱动的方法，用于获得 P411 酶催化的对映体选择性酶促卡宾 N-H 插入反应。通过计算建模，我们合理地引导工程努力，通过安装新的氢键锚固点来控制酶活性位点中关键内酯-卡宾（LAC）中间体的可及构象。这种氢键相互作用控制了反应性卡宾中间体的相对取向，使其能够对胺底物进行对映选择性的 N-亲核进攻。通过将 MD 模拟与定点饱和突变和筛选相结合，仅针对两个关键残基进行，我们能够反转以前工程化的 S 选择性 P411 酶的立体选择性。所得变体 L5_FL-B3 能够接受广泛的胺底物进行 N-H 插入，具有优异的产率（高达 >99%）、高效率（高达 12300 TTN）和良好的对映体控制（高达 7:93 er）。
