Is the acid/base catalytic residue mutation in β-D-mannosidase DtMan from Dictyoglomus thermophilum sufficient enough to provide thioglycoligase activity?
Is the acid/base catalytic residue mutation in β-D-mannosidase DtMan from Dictyoglomus thermophilum sufficient enough to provide thioglycoligase activity?
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DOI：
10.1016/j.biochi.2017.03.020
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发表时间：
2017-06-01
期刊：
影响因子：
3.9
通讯作者：
Daniellou, Richard
中科院分区：
文献类型：
--
作者：
Guillotin, Laure;Richet, Nicolas;Daniellou, Richard
糖苷水解酶可以通过酸/碱催化羧酸残基的突变转化为硫代糖基化酶。这些突变体已被证明对S糖苷的产生有价值，然而，文献中很少有实例描述有效的硫代糖苷酸酶活性，更少的是其潜在的分子机制。DtMan是嗜热Dictyoglomus thermophilum的GH2家族β-D-甘露糖苷酶，克隆并在E.coll.该重组蛋白对β-D-甘露糖苷具有高度专一性，并表现出与热稳定性相偶联的有效催化常数。然而，含有突变的酸/碱残基的7个变异体不能转化为有效的硫寡糖酶。DtMan Glu425Cys突变体的晶体结构和分子模拟计算表明，与已报道的其他GH2硫寡酶不同，硫醇受体的活性部位可及性可能会受到进入环刚性的影响。这一结构特征可能解释了为什么DtMan突变体没有显示出硫代糖酵解酶活性。(C)2017年Elsevier B.V.和法国兴业银行(SFBBM)。版权所有。
Glycoside hydrolases can be turned into thioglycoligase by mutation of the acid/base catalytic carboxylate residue. These mutants have proven valuable to generate S-glycosides, however, few examples in literature have described efficient thioglycoligase activity, and even fewer the underlying molecular mechanism. DtMan, a GH2 family beta-D-mannosidase from the thermophilic Dictyoglomus thermophilum was cloned and expressed in E. coll. The recombinant protein is highly specific for beta-D-mannosides, and exhibits efficient catalysis constants coupled to thermostability. However, seven variants bearing mutated acid/base residue could not be turned into efficient thioligases. Crystal structure of DtMan Glu425Cys mutant and molecular modeling calculations have demonstrated that unlike other GH2 thioligase reported, active site accessibility of thiol acceptor may be impaired by entrance loop rigidity. This structural feature may explain why DtMan mutants do not exhibit thioglycoligase activity. (C) 2017 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.