亚热带山区土壤未培养微生物L-赖氨酸脱羧酶Ldc1E中关键氨基酸残基的功能鉴定  

Functional Identification of the Key Amino Acids in a Metagenome-Derived L-lysine Decarboxylase Ldc1E from Subtropical Mountainous Soil Samples

作　　者：徐悦 邓洁 莫雪艳 欧倩 武波 蒋承建 Xu Yue;Deng Jie;Mo Xueyan;Ou Qian;Wu Bo;Jiang Chengjian(State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources,College of Life Science and Technology,Guangxi University,Nanning,530004)

机构地区：[1]广西大学生命科学与技术学院,亚热带农业生物资源保护与利用国家重点实验室,南宁530004

出　　处：《基因组学与应用生物学》2020年第6期2614-2620,共7页Genomics and Applied Biology

基　　金：国家自然科学基金项目(项目编号:31760437);广西自然科学基金项目(项目编号:2018JJA130051,2017JJB130020)共同资助。

摘　　要：本研究旨在探讨L-赖氨酸脱羧酶Ldc1E关键氨基酸在底物识别和催化过程中的作用;通过生物信息学方法选择突变位点,并利用直接定点突变技术,完成了6个关键氨基酸残基突变和功能鉴定研究。突变酶D692N最适温度和pH值分别为40℃和6.5。突变酶D692N比野生型Ldc1E对高温具有更强的耐受性,在40℃~55℃温浴1 h后剩余酶活力达到35%以上,在60℃温浴1 h后仍然保留20%的酶活力;而野生型酶Ldc1E在50℃以上温浴1 h后几乎失活。此外,50 mmol/L DMSO、5 mmol/L Al3+和Ca2+对突变酶的酶活力有激活作用,而Al3+对野生型酶Ldc1E具有明显抑制作用。突变酶D692N的分子动力学常数Km升高了1.78倍,kcat下降了20.2倍。突变酶S221A、H245A、D330A、H366A、F607Y经检测酶催化活性丧失。研究结果表明氨基酸残基位点D692对酶与底物的结合具有重要影响;而S221、H245、D330、H366、F607是Ldc1E酶活性能够体现的关键氨基酸位点,不可替换。本研究为探究L-赖氨酸脱羧酶的结构与功能关系提供理论参考。The aim of this study was to investigate the function of the key residues in the binding site of Ldc1E.Site-directed mutagenesis sites were identified based on the bioinformatics analysis. Furthermore, the mutation and functional identification of six residues were completed by site-directed mutagenesis method. The optimum temperature and pH value of mutant D692N were 40℃ and 6.5, respectively. The D692N mutant had higher temperature tolerance with remain of more than 35% of enzymatic activity after being incubated at 40℃~55℃ for1 h, and 20% enzymatic activity remained after being incubated at 60℃ for 1 h, while compared to the wild type the remaining activity is undetectable when the incubation temperature is above 60℃. In addition, the 50 mmol/L DMSO, 5 mmol/L Al3+ and Ca2+ were positive to the specific activity of mutant D692N, whereas Al3+ was negative to the wild-type Ldc1E. The Km values of mutant D692N were 1.78 folds higher than that of Ldc1E, however the kcat values were 20.2 folds lower than the wild type. While the mutant of S221A, H245A, D330A, H366A, F607Y had been demonstrated that lost the whole activity. The results demonstrated that the residue D692 had an important influence on the binding activity between the enzyme and the substrate, and the amino acid residues S221, H245, D330, H366, F607 were key amino acid residues to the enzymatic activity of Ldc1E. This research provided theory references to the structure-function of L-lysine decarboxylase.

关 键 词：L-赖氨酸脱羧酶 定点突变 同源建模 分子对接 结构和功能 

分 类 号：S154.3[农业科学—土壤学]