Crystal Structure And Active Site Analysis Of Cyclohexylamine Oxidase From Acinetobacter Sp.YT-02

Cyclohexylamine oxidase is an amine oxidase that catalyzes the formation of cyclohexanone from cyclohexylamine,and is widely used in the degradation of environmental pollutants and the chiral resolution of amine compound enantiomers.Acinetobacter sp.YT-02 is a bacterium isolated by our laboratory that can grow with cyclohexylamine as the sole carbon and nitrogen sources.The cyclohexylamine oxidase(CHAO)from bacterium YT-02 has been heterologously expressed,purified and characterized.The results showed that its activity was nearly 162 times higher than that of CHAO_IH-35A,but its catalytic mechanism is unclear.The main purpose of this study is to obtain the crystals of cyclohexylamine oxidase CHAO_YT-02,analyze its crystal structure by X-ray crystallography,and identify key amino acid residues in its active center through experiments.In order to achieve this goal,the protein was purified with a nickel column and molecular sieve chromatography,and its concentration reached 6.147±0.087 mg/m L,and the purity of the protein exceeded 95%.The crystals were screened in the kit by sitting-drop meteorological diffusion method.The crystals precipitated in the solution of 0.1 M sodium citrate(p H 5.5)and22%PEG1000,the crystals were larger but thinner.We changed the p H of sodium citrate solution and the concentration of PEG1000 separately to further sieve the crystals.Finally,high-quality crystals that met the requirements of X-ray diffraction were obtained in the solution of 0.1 M sodium citrate(p H 5.5)and 20%PEG1000.Then,crystals in cryoprotectant were stored in liquid nitrogen.The diffraction data were collected at Shanghai synchrotron radiation facility.After data processing by XDS software,the structure was determined at 1.49?by molecular replacement method using structure of CHAO_IH-35A as a template.The amino acid residues at the substrate binding site(Leu302,Trp70,Phe197,Phe349,and Tyr440)and the intermediate cavity site(Ile180,Leu181,Ile208,and Trp332)are predicted to be the key amino acid residues at the active center.The mutants in which nine amino acid residues were mutated into Ala,respectively,were successfully constructed with one-step cloning technique and overlapping PCR technique.Furthermore,mutants Trp70Ala,Ile180Ala,Leu181Ala and Ile208Ala were expressed and purified after the induction,and the activity of these four mutants were measured.The results showed that Trp70Ala and wild-type protein have the same substrate profile,but its activity is only equivalent to 0.01%of that of wild-type,indicating Trp70 present in substrate binding site,consistent with crystal structure-based analysis results;Ile180Ala,Leu181Ala,and Ile208Ala all lost activity against the substrate cyclohexylamine,and the purified mutants Ile180Ala and Leu181Ala were both colorless,which is inconsistent with the yellow color of wild-type capable of binding to FAD,indicating that Ile180 and Leu181 may be involved in binding to cofactor FAD.In this study,the crystal structure of CHAO_YT-02 was determined,the active sites were obtained,and mutants at 9 different active sites were constructed,but only 4 active sites in the mutants were verified,and the remaining 5 sites are yet to be tested in subsequent experiments.It lays the foundation for elucidating the catalytic mechanism of CHAO_YT-02 from a structural perspective,and also provides a theoretical basis for the directed evolution of CHAO_YT-02.