Gao Weihua, Dai Sen, Liu Quanli, Xu Haijin, Bai Yanlin, Qiao Mingqiang
The Key Laboratory of Molecular Microbiology and Technology, Ministry of Education of China, 94 Weijin Road, College of Life Sciences, Nankai University, Tianjin 300071, China.
Mikrobiologiia. 2010 Nov-Dec;79(6):774-8.
Bacillus subtilis aconitase, encoded by the citB gene, is a bifunctional enzyme, which can not only interconvert citrate and isocitrate, but also has the RNA binding function similar to the eukaryotic protein IRP-1 (iron regulatory protein 1). Homology analysis between eukaryotic aconitase and B. subtilis aconitase indicates that the amino acids 741-745 probably have important function for the B. subtilis aconitase. To analyse the exact effect of these amino acids for aconitase activity, a site-directed mutagenesis of the citB is constructed, in which, the Arg741 and Gln745 are both changed into Glu. The resulting strain exhibits an increased enzymatic activity of aconitase comparing to that of the wild-type strain. Western blotting shows that the aconitase protein expression level is significantly increased in the mutant strain. By beta-galactosidase activity assay, the transcription level of citB is also increased. These results indicate that the mutation of citB gene has significant effect on B. subtilis aconitase transcription, expression and enzymatic activity.
由citB基因编码的枯草芽孢杆菌乌头酸酶是一种双功能酶，它不仅可以使柠檬酸和异柠檬酸相互转化，还具有与真核蛋白IRP-1（铁调节蛋白1）相似的RNA结合功能。真核乌头酸酶与枯草芽孢杆菌乌头酸酶之间的同源性分析表明，第741-745位氨基酸可能对枯草芽孢杆菌乌头酸酶具有重要功能。为了分析这些氨基酸对乌头酸酶活性的确切影响，构建了citB的定点突变体，其中，第741位的精氨酸和第745位的谷氨酰胺都被替换为谷氨酸。与野生型菌株相比，所得菌株的乌头酸酶活性有所增加。蛋白质免疫印迹显示，突变菌株中乌头酸酶蛋白表达水平显著增加。通过β-半乳糖苷酶活性测定，citB的转录水平也有所增加。这些结果表明，citB基因的突变对枯草芽孢杆菌乌头酸酶的转录、表达和酶活性具有显著影响。
