Analysis reaction mechanism of ACC deaminase by X-ray crystallography

点击翻译按钮获取中文摘要

To consider the role of the reaction of the 1-Aminocyclopropane-1-carboxylate Deaminase (ACCD), except from residues Lys51 and Lys54, Ser78 and Ans79 were chosen for site-directed mutagenesis basis on the structure of yeast ACCD (yACCD). In this year, we have got success for overexpression of K51A, K51T, K54A, S78A, N79A, N79D and N79S mutants, since expression vector was changed. Apart from K51A and K54A, all mutants were purified.Because ACCD is PLP-dependent enzyme, analysis active can be carried out only by absorbance spactra for purified mutants of K51T, S78A, N79A, N79D and N79S.The results shown that all of them bound co-enzyme and were inactivation with substrate ACC.Now, crystallization of K51T mutant is in progress.On the other hand, hyperthermophilic arcaebacteria Pyrococcus horikoshii OT3 that all sequence were provided by the genome sequencing projects, has a protein which was assigned to ACCD (phoACCD) and conserves residues around active center comparison with yACCD.However, this enzyme shows active with substrate L-Ala and L-Ser, but not ACC.The structure of phoACCD may give important information for understanding the active mechanism of ACCD.We have express phoACCD in E.coli. and purified it. The crystals of phoACCD were grown up at room temperature and diffracted to 2.2Å resolution. The crystal structure analysis is in progress.