人工酶创制用于手性酮类化合物的生物合成

Artificial enzyme design for the biosynthesis of chiral ketones

摘要:

手性酮类化合物的高效不对称合成是绿色生物制造的重要挑战。为解决天然仲胺酶稀缺及现有人工酶对酮类底物活化能力不足的问题，本研究基于乳酸乳球菌多药耐药调节因子(LmrR)蛋白骨架，通过二硫键共价修饰策略，将含有芳香仲胺中心的异吲哚啉(isoindoline, IIDN)引入其疏水空腔，构建了一种新型人工仲胺酶LmrR_V99C_IIDN。该酶能够通过烯胺催化机制活化不饱和烯酮底物，催化其与丙二酸二苄酯的不对称迈克尔加成反应。经过位点筛选与定向进化，获得最优突变体LmrR_V99C_N19Y_IIDN，其对环己烯酮的催化反应产率达73%，对映选择性为65%。底物拓展研究表明，该酶对环状烯酮(如环己烯酮、环戊烯酮)表现出良好催化活性与立体选择性，而对链状烯酮的催化效率较低。本研究为酮类化合物的不对称功能化提供了新型生物催化工具，并展示了非天然催化中心与蛋白质骨架协同调控反应选择性的潜力，为拓展人工酶在复杂手性分子合成中的应用奠定了基础。

Abstract:

Efficient asymmetric synthesis of chiral ketones is a significant challenge in green biomanufacturing. To address the scarcity of natural secondary amine enzymes and the insufficient activation capability of existing artificial enzymes towards ketone substrates, this study developed a novel artificial secondary amine enzyme, LmrR_V99C_IIDN, based on the protein scaffold of the Lactococcus lactis multidrug resistance regulator (LmrR). A covalent modification strategy using a disulfide bond was employed to introduce an isoindoline (IIDN) moiety containing an aromatic secondary amine center into the hydrophobic cavity of the protein. This engineered enzyme activates unsaturated enone substrates via an enamine catalysis mechanism, catalyzing their asymmetric Michael addition with dibenzyl malonate. Through site-specific screening and directed evolution, the optimal mutant LmrR_V99C_N19Y_IIDN was obtained. This variant achieved a yield of 73% and an enantioselectivity of 65% in the catalytic reaction with cyclohexenone. Substrate scope studies demonstrated that the enzyme exhibited good catalytic activity and stereoselectivity towards cyclic enones (such as cyclohexenone and cyclopentenone), while showing lower catalytic efficiency for linear enones. This study provides a new biocatalytic tool for the asymmetric functionalization of ketones and highlights the potential of synergistically regulating reaction selectivity through the combination of non-natural catalytic centers and protein scaffolds. It lays a foundation for expanding the application of artificial enzymes in the synthesis of complex chiral molecules.