| Alcohol dehydrogenases,as one of the most important catalysts of metabolic species in organisms,are widely used in biocatalysis and biomedicine and other fields.The exploration of the complete internal structure of the alcohol dehydrogenase(ADH)active site is critical because of its conformational changes and more complex reaction system.The6-carboxyhexanol dehydrogenase(ChnD),derived from Acinetobacter NICMB 9871 strain,is one of the key enzymes in the process of metabolism of cyclohexanol,which catalyzes the oxidative dehydrogenation of 6-carboxyhexanol to 6-carboxyhexanal and finally produces metabolites such as adipic acid,acetyl Co A and succinyl Co A,which have important roles in industry,food,medicine as well as biological research.In order to explore the catalytic mechanism of ChnD,in this study,we tried to solve the structure of ChnD and its substrate-bound complex structure by crystallography,and illustrate its catalytic mechanism.In this study,the gene of ChnD was cloned into the pRSFDuet-1 vector for high expression in Escherichia coli.The resulting protein was purified with a nickel column coupled with an ion exchange column purification system.Because of the protein aggregate,we obtained protein optimum buffer using dynamic light scattering test,the best buffer is 20 mM Tris,pH 8.5 and 20 mM HEPES,500 mM NaCl,pH 7.5.We obtained ChnD and its co crystal with substrate and coenzyme by using the sitting drop method for crystallization.The crystal shape and size were improved by optimizing the seed method,and better crystals were selected for X-ray diffraction.In this study,we constructed a highly efficient expression system of ChnD and obtained a large amount of target proteins with high purity.A large number of protein crystals were obtained by co-crystallization with substrate and coenzyme,and the X-ray diffraction data were collected,which laid a solid foundation for further analysis of the structure of ChnD. |
PDF Full Text Request