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

[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.