Crystal structure of cyclohexylamine oxidase from Acinetobacter sp. YT−02 reveals key residues for catalytic activity and substrate specificity

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_YT-02 indicated that its specific activity towards cyclohexylamine of CHAO_YT-02 was ten times higher than that of its homolog CHAO_IH-35A. In this study, the crystal structure of CHAO_YT-02 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_YT-02 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_YT-02. This study on the structure and catalytic mechanism of cyclohexylamine oxidase is beneficial to eliminating toxic amine compounds in the environment.