氧化型辅酶NAD^+及布氏杆菌SahH活性位点对SahH催化活性的影响  

Effects of oxidized coenzyme NAD^+and SahH active site on catalytic activity of Brucella SahH

作　　者：荆雅玮 左佳坤 王志豪 胡剑刚 黄燕[1] 米荣升[1] 苗晋锋[2] PHOUTHAPANE Vanhnaseng 陈兆国[1] 韩先干[1] JING Yawei;ZUO Jiakun;WANG Zhihao;HU Jiangang;HUANG Yan;MI Rongsheng;MIAO Jinfeng;PHOUTHAPANE Vanhnaseng;CHEN Zhaoguo;HAN Xiangan(Shanghai Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Shanghai 200241,China;College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;Animal Science Center at Biotechnology and Ecology Institute,Ministry of Science and Technology of Laos,Vientiane 22797,Laos)

机构地区：[1]中国农业科学院上海兽医研究所,上海200241 [2]南京农业大学动物医学院,江苏南京210095 [3]老挝科技部生态与生物技术研究所,万象22797

出　　处：《南京农业大学学报》2020年第5期903-909,共7页Journal of Nanjing Agricultural University

基　　金：国家自然科学基金项目(31572546,31872483);国家重点研发计划专项(2018YFE0102200);上海市科技兴农重点攻关项目(2019-02-08-00-08-F01151)。

摘　　要：[目的]本文旨在鉴定影响布氏杆菌S-腺苷同型半胱氨酸水解酶(S-adenosylhomocysteine hydrolase,SahH)催化S-腺苷同型半胱氨酸(S-adenosylhomocysteine,SAH)产生同型半胱氨酸(homocysteine,HCY)的催化活性位点。[方法]将表达载体pET28a-Bru-sahH转化大肠杆菌BL21中,表达、纯化布氏杆菌SahH(Bru-SahH)蛋白,分析辅酶NAD+、磷酸化修饰及活性位点对Bru-SahH催化活性的影响。[结果]添加NAD+后,Bru-SahH的催化活性降低85.6%;对Bru-SahH质谱分析表明,其444位丝氨酸为可能的磷酸化位点,对该位点突变后Bru-SahH催化SAH形成HCY的活性降低85.5%;生物信息学分析表明,Bru-SahH的337位和338位氨基酸为其活性位点,分别对上述2个位点进行单突变和双突变,突变后的Bru-SahH催化活性降低90.8%以上。[结论]Bru-SahH的337、338和444位氨基酸是其催化活性位点,添加外源性NAD+可抑制其催化活性。[Objectives]The objective of this paper is to investigate the influencing factors of catalytic activity and identification of active catalytic site of Brucella S-adenosylhomocysteine hydrolase(SahH).[Methods]The expression vector pET28a-Bru-sahH was transformed into Escherich coli BL21,the influencing factors of catalytic activity of SahH in vitro were analyzed by addition of exogenous coenzyme NAD+,analysis of modification and identification of catalytic activity sites of SahH,respectively.[Results]The results showed that the catalytic activity of recombinant Brucella protein SahH(Bru-SahH)was decreased by 85.6%by addition of exogenous NAD+.Furthermore,the catalytic activity of Bru-SahH was decreased by 85.5%by mutation of 444th amino acid,which was a possible phosphorylation modification by mass spectrum analysis of Bru-SahH.In addition,the catalytic activity of Bru-SahH was decreased more than 90.8%by single or double site mutation at 337th and/or 338th amino acids of Bru-SahH,which was predicted to the key activity sites of Bru-SahH by bioinformatics analysis.[Conclusions]The results indicated that the key catalytic sites of Bru-SahH were 337th,338th and 444th amino acids.Exogenous NAD+could inhibit catalytic activity of Bru-SahH.

关 键 词：布氏杆菌 S-腺苷同型半胱氨酸水解酶 酶活性 活性位点 

分 类 号：S855.12[农业科学—临床兽医学]