Wu Jing, Han Zhenggang, Li Pengrong, Li Jing, Chen Yuanyuan, Ning Shangbo, Chao Hong-Jun, Gao Xue-Wang, Yan Dazhong
School of Life Science and Technology, Wuhan Polytechnic University, Wuhan, 430023, PR China.
Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, 100190 PR China.
Enzyme Microb Technol. 2025 Oct;190:110700. doi: 10.1016/j.enzmictec.2025.110700. Epub 2025 Jun 23.
Cyclohexylamine oxidase is a member of amine oxidases that catalyzes the conversion of cyclohexylamine to cyclohexanone. In our previous work, the enzymatic activity assay of cyclohexylamine oxidase CHAO indicated that its specific activity towards cyclohexylamine of CHAO was ten times higher than that of its homolog CHAO. In this study, the crystal structure of CHAO was determined by the molecular replacement method at a resolution of 1.49 Å. The atomic structure revealed that the amino acid residues Leu302, Trp70, Phe197, Phe349, and Tyr440 constitute the active center pocket of the enzyme. Amino acid residues Ile180, Leu181, and Trp332 separate the active center pocket and an intermediate pocket. Moreover, a molecular dynamics (MD) simulation and the calculation of the binding free energy were performed to predict substrate entry and product release from cyclohexylamine oxidases. Single-amino acid substitution mutants (W70A, I180A, L181A, I208A, F197A, L302A, W332A, F349A, and Y440A) of CHAO were constructed to investigate the role of these amino acid residues in enzymatic properties and substrate specificity. The results indicated that both the amino acid residues in the active center pocket and gating the two pockets affected the activity or substrate specificity of CHAO. This study on the structure and catalytic mechanism of cyclohexylamine oxidase is beneficial to eliminating toxic amine compounds in the environment.
环己胺氧化酶是胺氧化酶家族的一员，可催化环己胺转化为环己酮。在我们之前的工作中，环己胺氧化酶CHAO的酶活性测定表明，其对环己胺的比活性是其同源物CHAO的十倍。在本研究中，通过分子置换法以1.49 Å的分辨率测定了CHAO的晶体结构。原子结构显示，氨基酸残基Leu302、Trp70、Phe197、Phe349和Tyr440构成了该酶的活性中心口袋。氨基酸残基Ile180、Leu181和Trp332将活性中心口袋与一个中间口袋分隔开。此外，进行了分子动力学（MD）模拟和结合自由能计算，以预测环己胺氧化酶的底物进入和产物释放。构建了CHAO的单氨基酸取代突变体（W70A、I180A、L181A、I208A、F197A、L302A、W332A、F349A和Y440A），以研究这些氨基酸残基在酶性质和底物特异性中的作用。结果表明，活性中心口袋中的氨基酸残基以及分隔两个口袋的结构都影响了CHAO的活性或底物特异性。这项关于环己胺氧化酶结构和催化机制的研究有助于消除环境中的有毒胺类化合物。
