Characterization of the catalytic disulfide bond in E. coli 4-thiouridine synthetase to elucidate its functional quaternary structure.
Characterization of the catalytic disulfide bond in E. coli 4-thiouridine synthetase to elucidate its functional quaternary structure.
复制标题
大肠杆菌 4-硫尿苷合成酶中催化二硫键的表征，以阐明其功能性四级结构。
DOI：
10.1002/pro.2965
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发表时间：
2016
期刊：
影响因子：
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通讯作者：
Mueller,EugeneG
中科院分区：
文献类型：
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作者：
Veerareddygari,GovardhanReddy;Klusman,ThomasC;Mueller,EugeneG
在原核tRNA的第8位的4-硫代尿苷作为近紫外光的光传感器，尿苷到4-硫代尿苷的转录后转化由4-硫代尿苷合成酶（s4 US，也称为ThiI）催化，其分为两类，不同之处在于C末端罗丹同源结构域的存在。该结构域中的半胱氨酸残基首先带有过硫化物基团，然后与两类s4 US中保守的半胱氨酸残基形成二硫键。最近的晶体结构表明，s4 US在RNA底物的存在下二聚化，每个亚基的结构域有助于一个RNA分子的结合和反应，这就提出了一个问题，即在较长的s4 US类中的催化二硫键是否在亚基内或亚基之间形成。你.大肠杆菌酶是s4 US的较长类别中表征最好的成员，并且在单次催化转换期间定量安装二硫键后对其进行了检查。凝胶电泳和蛋白水解/MALDI-MS结果强烈暗示，二硫键在单个亚基内形成，这为具有附加的罗丹岛同源结构域的s4 US类的结构建模以及实验的设计和解释提供了重要的约束，以探测催化过程中结构域的动力学。
4‐Thiouridine at position 8 in prokaryotic tRNA serves as a photosensor for near‐UV light, and the posttranscriptional conversion of uridine to 4‐thiouridine is catalyzed by the 4‐thiouridine synthetases (s4US, also named ThiI), which fall into two classes that differ in the presence of a C‐terminal rhodanese homology domain. A cysteine residue in this domain first bears a persulfide group and then forms a disulfide bond with a cysteine residue that is conserved in both classes of s4US. Recent crystal structures suggest that s4US dimerizes in the presence of RNA substrate with domains from each subunit contributing to the binding and reaction of one RNA molecule, which raises the question of whether the catalytic disulfide bond in the longer class of s4US is formed within or between subunits. TheE. colienzyme is the best‐characterized member of the longer class of s4US, and it was examined after quantitative installation of the disulfide bond during a single catalytic turnover. Gel electrophoresis and proteolysis/MALDI‐MS results strongly imply that the disulfide bond forms within a single subunit, which provides a vital constraint for the structural modeling of the class of s4US with an appended rhodanese homology domain and the design and interpretation of experiments to probe the dynamics of the domains during catalysis.