摘要
非解朊栖热菌HG10 2耐热 β_糖苷酶为 (β α) 8桶状结构 ,是具有水解功能和转糖苷功能的单体酶。该酶可以作为一个很好的模型来研究糖苷酶的反应机制、底物特异性和耐热的分子基础。根据对该酶的晶体结构解析和同家族酶的结构比较 ,推测Glu16 4和Glu338分别是质子供体和亲核基团两个活性位点 ;在α_螺旋N端第一位的脯氨酸和蛋白质外周的精氨酸是耐热机制的关键位点和关键氨基酸残基。为确定这些氨基酸残基的功能 ,通过基因定点突变的方法分别把Glu16 4、Glu338、Pro316、Pro35 6、Pro344和Arg32 5置换成Gln、Ala、Gly、Ala、Phe和Leu ,同时还对Pro316和Pro35 6进行了双置换。突变酶经过纯化得到电泳纯 ,用CD光谱进行了野生酶和突变酶的结构比较。通过突变酶的酶功能和酶学性质分析 ,结果表明Glu16 4和Glu338分别是质子供体和亲核基团 ,亲核基团的突变酶TnglyE338A可以合成混合型糖苷键寡糖类似物 ;在α_螺旋N端第一位的Pro316和Pro35 6以及在蛋白质外周形成离子键的Arg32 5均是对耐热性有贡献的关键氨基酸残基。
Glycosidase (Tngly) from the thermophilic eubacterium Thermus nonproteolyticus HG102, which is a thermostable monomeric protein and adopts the (β/α) 8 barrel fold, is an excellent model system to be investigated for the thermostable mechanism, activity and substrate specificity. Here, based on the analysis of structural basis for thermostability of Tngly (Wang et al, 2003) and comparison of other proteins structure of homofamily, Glu164 and Glu338 may act as proton donor and nucleophile in the hydrolysis reaction respectively; proline located at N1 of α_helix and arginine which can form ion link may contribute to the thermostability. We aim to further identify the critical sites and the amino acid residue(s) responsible for the activity, the thermal stability and the substrate specificity. Mutations had been constructed by site_directed mutagenesis. They are Glu164Gln, Glu338Ala, Pro316Gly, Arg325Leu, Pro344Phe, Pro356Ala and Pro316Gly/Pro356Ala. All mutant proteins were purified to SDS PAGE purity. Changes in the conformations were examined by means of CD. The Glu338Ala mutant showed no detectable hydrolysis activity, but can synthesize oligosaccharides, as expected for the residue acting as the nucleophile of the reaction. The Glu164 acts as the general acid/base catalyst in the hydrolysis reaction. Changes in stabilities of mutants compared with wild_type were determined by means of heat inactivity experiment. These results indicate that the amino acid residue of proline that is located at N1 positions of α_helix, and Arg325 that form salt bridge between α_helices 5 and α_helices 6, are the critical sites to protein thermostabilization.
出处
《生物工程学报》
CAS
CSCD
北大核心
2005年第1期84-91,共8页
Chinese Journal of Biotechnology
基金
中国科学院知识创新工程项目基金资助 (No .0 10 3 )。~~
关键词
β-糖苷酶
转糖苷活性
热稳定性
定点突变
β-glycosidase, transglycosylation, thermostability, site-directed mutagenesis