Karagüler Nevin Gül, Sessions Richard B, Clarke Anthony R
Department of Molecular Biology and Genetics, Faculty of Science and Letters, Istanbul Technical University, Istanbul, Turkey.
Biotechnol Lett. 2007 Sep;29(9):1375-80. doi: 10.1007/s10529-007-9392-8. Epub 2007 May 4.
Wild-type cmFDH contains no cystines, hence it is a good candidate to test the hypothesis that thermostability can be achieved by introducing new disulphide bridges. Three cysteine double mutants of cmFDH were designed, using a homology model reported previously, to introduce cystine bridges in the C-domain (T169C-T226C) in the N-domain (V88C-V112C) and between the two monomers (M156C-L159C) to form two cystine bridges across the dimer interface. These mutants were constructed and the proteins were over-expressed in E. coli. The mutants V88C-V112C and M156C-L159C lost FDH activity. The mutant T169C-T226C was both less active and less thermostable than wild-type FDH.
野生型甲酸脱氢酶（cmFDH）不含胱氨酸，因此它是检验通过引入新的二硫键来实现热稳定性这一假说的理想候选对象。利用先前报道的同源模型，设计了cmFDH的三个半胱氨酸双突变体，以便在C结构域（T169C-T226C）、N结构域（V88C-V112C）以及两个单体之间（M156C-L159C）引入胱氨酸桥，从而在二聚体界面形成两个二硫键桥。构建了这些突变体，并在大肠杆菌中对蛋白质进行了过量表达。突变体V88C-V112C和M156C-L159C失去了甲酸脱氢酶活性。突变体T169C-T226C的活性和热稳定性均低于野生型甲酸脱氢酶。
