Structural basis for catalytic activity of a silkworm Delta-class glutathione transferase.
Structural basis for catalytic activity of a silkworm Delta-class glutathione transferase.
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DOI：
10.1016/j.bbagen.2012.04.022
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发表时间：
2012-10
期刊：
影响因子：
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通讯作者：
Kohji Yamamoto;K. Usuda;Y. Kakuta;M. Kimura;A. Higashiura;A. Nakagawa;Y. Aso;Mamoru Suzuki
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文献类型：
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作者：
Kohji Yamamoto;K. Usuda;Y. Kakuta;M. Kimura;A. Higashiura;A. Nakagawa;Y. Aso;Mamoru Suzuki
背景谷胱甘肽转移酶(GST)催化谷胱甘肽结合，是外源物质和内源性物质的主要解毒途径。在这里，我们测定了家蚕Delta类GST(BmGSTD)的晶体结构，以检测其催化残基。方法用分子置换方法解析bmGSTD的三维结构，并将其分辨率提高到2.0？结果与冈比亚按蚊Delta类GST的结构比对表明，bmGSTD包含两个不同的结构域(N-末端结构域和C-末端结构域)。结合的谷胱甘肽定位于N-末端结构域。通过定点突变将可能的催化残基转化为丙氨酸，并用谷胱甘肽和1-氯-2，4-二硝基苯(GST的合成底物)对突变体的催化活性进行了表征。对bmGSTD突变体的动力学分析表明，Ser11、Gln51、His52、Ser67和Arg68对酶的功能是重要的。这些结果为bmGSTD催化家蚕谷胱甘肽结合提供了结构信息。
BACKGROUNDGlutathione transferase (GST) catalyzes glutathione conjugation, a major detoxification pathway for xenobiotics and endogenous substances. Here, we determined the crystal structure of a Delta-class GST from Bombyx mori (bmGSTD) to examine its catalytic residues.METHODSThe three-dimensional structure of bmGSTD was resolved by the molecular replacement method and refined to a resolution of 2.0Å.RESULTSStructural alignment with a Delta-class GST of Anopheles gambiae indicated that bmGSTD contains 2 distinct domains (an N-terminal domain and a C-terminal domain) connected by a linker. The bound glutathione localized at the N-terminal domain. Putative catalytic residues were changed to alanine by site-directed mutagenesis, and the resulting mutants were characterized in terms of catalytic activity using glutathione and 1-chloro-2,4-dinitrobenzene, a synthetic substrate of GST. Kinetic analysis of bmGSTD mutants indicated that Ser11, Gln51, His52, Ser67, and Arg68 are important for enzyme function.GENERAL SIGNIFICANCEThese results provide structural insights into the catalysis of glutathione conjugation in B. mori by bmGSTD.