Structural basis for the enhanced thermal stability of alcohol dehydrogenase mutants from the mesophilic bacterium Clostridium beijerinckii:: contribution of salt bridging
Structural basis for the enhanced thermal stability of alcohol dehydrogenase mutants from the mesophilic bacterium Clostridium beijerinckii:: contribution of salt bridging
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DOI：
10.1110/ps.0222102
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发表时间：
2002-11-01
期刊：
影响因子：
8
通讯作者：
Burstein, Y
中科院分区：
文献类型：
--
作者：
Bogin, O;Levin, I;Burstein, Y
我们实验室先前的研究比较了两种高度同源的醇脱氢酶的三维结构元件，一种来自嗜温菌拜氏梭菌（CbADH），另一种来自极端嗜热菌嗜热厌氧杆菌（TbADH），表明在嗜热酶中，亚基间界面处的额外亚基内离子对（Glu 224-Lys 254）和短离子对网络（Lys 257-Asp 237-Arg 304-Glu 165）可能有助于TbADH的极端热稳定性。在本研究中，我们使用定点突变，以取代这些结构上的战略性残基在CbADH与相应的氨基酸从TbADH，我们确定了这种替代对CbADH的热稳定性的影响。亚基内离子对区域的突变增加了单突变体S254 K-和双突变体V224 E/S254 K-CbADH的热稳定性，但不增加单突变体V224 E-CbADH的热稳定性。两个单一的氨基酸置换，M304 R-和Q165 E-CbADH，在该地区的亚基间离子对网络增强热稳定性，在双突变M304 R/Q165 E-CbADH的累加效应。为了研究这种突变改变CbADH分子结构以实现增强的热稳定性的确切机制，我们构建了四重突变体V224 E/S254 K/Q165 E/M304 R-CbADH并解析其三维结构。总体结果表明，在CbADH突变体中的氨基酸取代增强热稳定性，通过形成由水分子介导的亚基间离子对和盐桥的扩展网络以及通过形成新的亚基内盐桥来增强酶的四级结构。
Previous research in our laboratory comparing the three-dimensional structural elements of two highly homologous alcohol dehydrogenases, one from the mesophile Clostridium beijerinckii (CbADH) and the other from the extreme thermophile Thermoanaerobacter brockii (TbADH), suggested that in the thermophilic enzyme, an extra intrasubunit ion pair (Glu224-Lys254) and a short ion-pair network (Lys257-Asp237-Arg304-Glu165) at the intersubunit interface might contribute to the extreme thermal stability of TbADH. In the present study, we used site-directed mutagenesis to replace these structurally strategic residues in CbADH with the corresponding amino acids from TbADH, and we determined the effect of such replacements on the thermal stability of CbADH. Mutations in the intrasubunit ion pair region increased thermostability in the single mutant S254K- and in the double mutant V224E/S254K-CbADH, but not in the single mutant V224E-CbADH. Both single amino acid replacements, M304R- and Q165E-CbADH, in the region of the intersubunit ion pair network augmented thermal stability, with an additive effect in the double mutant M304R/Q165E-CbADH. To investigate the precise mechanism by which such mutations alter the molecular structure of CbADH to achieve enhanced thermostability, we constructed a quadruple mutant V224E/S254K/Q165E/M304R-CbADH and solved its three-dimensional structure. The overall results indicate that the amino acid substitutions in CbADH mutants with enhanced thermal stability reinforce the quaternary structure of the enzyme by formation of an extended network of intersubunit ion pairs and salt bridges, mediated by water molecules, and by forming a new intrasubunit salt bridge.