Analysis reaction mechanism of ACC deaminase by X-ray crystallography

为了考虑1-氨基环丙烷-1-羧酸脱氨酶（ACCD）反应的作用，除Lys51和Lys54残基外，根据酵母ACCD （yACCD）的结构选择Ser78和Ans79进行定点诱变。在这一年里，我们成功过表达了K51A、K51T、K54A、S78A、N79A、N79D和N79S突变体，因为我们改变了表达载体。除K51A和K54A外，其余突变体均被纯化。由于ACCD是plp依赖性酶，因此对纯化的K51T、S78A、N79A、N79D和N79S突变体只能通过吸光度空间进行活性分析。结果表明，它们均与辅酶结合，并与底物ACC失活。现在，K51T突变体的结晶正在进行中。另一方面，所有序列均由基因组测序项目提供的嗜热古细菌阔氏焦球菌（Pyrococcus horikoshii OT3），与yACCD相比，具有一个被分配到ACCD的蛋白（phoACCD），并且在活性中心周围保留残基。然而，该酶对底物L-Ala和L-Ser有活性，而对ACC无活性。phoACCD的结构为理解ACCD的作用机制提供了重要的信息。我们在大肠杆菌中表达了phoACCD。然后净化它。在室温下生长phoACCD晶体并衍射至2.2Å分辨率。晶体结构分析正在进行中。

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.