College of Material, Chemistry and Chemical Engineering, Key Laboratory of Organosilicon Chemistry and Material Technology of Ministry of Education, Hangzhou Normal University, Hangzhou, 311121, China.
School of Basic Medical Sciences, Hangzhou Normal University, Hangzhou, 311121, China.
Appl Microbiol Biotechnol. 2023 Dec;107(23):7089-7104. doi: 10.1007/s00253-023-12767-y. Epub 2023 Sep 21.
Nitriles are of significant interest in the flavor and fragrance industries with potential application in cosmetics due to their higher stability than analogous aldehydes. However, the traditional methods to prepare nitriles need toxic reagents and hash conditions. This work aimed to develop a chemoenzymatic strategy to synthesize nitriles from natural aldehydes with aldoxime as the intermediate. A non-classical aldoxime dehydratase (Oxd) was discovered from the fungus Aspergillus ibericus (OxdAsp) to catalyze the dehydration of aldoximes to corresponding nitriles under mild conditions. The amino acid sequence of OxdAsp exhibits an approximately 20% identity with bacterial Oxds. OxdAsp contains a heme prosthetic group bound with the axial H287 in the catalytic pocket. The structure models of OxdAsp with substrates suggest that its catalytic triad is Y138-R141-E192, which is different from the classically bacterial Oxds of His-Arg-Ser/Thr. The catalytic mechanism of OxdAsp was proposed based on the mutagenesis of key residues. The hydroxyl group of the substrate is fixed by E192 to increase its basicity. Y138 acts as a general acid-based catalyst, and its phenolic proton is polarized by the adjacent R141. The protonated Y138 would donate a proton to the hydroxyl group of the substrate and eliminate a water molecule from aldoxime to produce nitrile. The recombinant OxdAsp can efficiently dehydrate citronellal oxime and cinnamaldoxime to citronellyl nitrile and cinnamonitrile in aqueous media, which are applied as fragrance ingredients in the food and cosmetic fields. KEY POINTS: • A novel aldoxime dehydratase from the Aspergillus genus was first characterized as a heme-binding protein. • The catalytic mechanism was predicted based on the molecular interactions of the catalytic pocket with the substrate. • A chemoenzymatic strategy was developed to synthesize nitriles from natural aldehydes with aldoxime as the intermediate.
腈类化合物在香精和香料行业中具有重要的应用价值，由于其稳定性高于相应的醛类，因此在化妆品行业也有潜在的应用。然而，传统的腈类化合物制备方法需要使用有毒试剂和苛刻的条件。本研究旨在开发一种从天然醛类化合物通过肟作为中间体合成腈类化合物的酶法策略。从真菌 Aspergillus ibericus 中发现了一种非经典的醛肟脱水酶（Oxd），该酶可以在温和条件下催化醛肟脱水生成相应的腈类化合物。OxdAsp 的氨基酸序列与细菌 Oxds 具有约 20%的同源性。OxdAsp 含有一个与催化口袋中轴向 H287 结合的血红素辅基。与底物的结构模型表明，其催化三联体为 Y138-R141-E192，与经典的细菌 Oxds 的 His-Arg-Ser/Thr 不同。基于关键残基的突变，提出了 OxdAsp 的催化机制。通过 E192 固定底物的羟基，增加其碱性。Y138 作为一个广义的酸催化剂，其酚质子被相邻的 R141 极化。质子化的 Y138 会向底物的羟基提供一个质子，并从醛肟中消除一个水分子，生成腈。重组 OxdAsp 可以在水相介质中高效地将柠檬醛肟和肉桂醛肟脱水生成柠檬腈和肉桂腈，这些腈类化合物作为香料成分应用于食品和化妆品领域。关键点：• 首次从曲霉属中鉴定出一种新型的醛肟脱水酶，其为血红素结合蛋白。• 根据催化口袋与底物的分子相互作用，预测了催化机制。• 开发了一种从天然醛类化合物通过肟作为中间体合成腈类化合物的酶法策略。
