title: Effect of site-directed mutagenesis of citB on the expression and activity of Bacillus subtilis aconitase.
abstract: 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.
authors: 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.
authors: Weihua
authors: W
authors: Gao
authors: Sen
authors: S
authors: Dai
authors: Quanli
authors: Q
authors: Liu
authors: Haijin
authors: H
authors: Xu
authors: Yanlin
authors: Y
authors: Bai
authors: Mingqiang
authors: M
authors: Qiao
mesh_terms: D000154:Aconitate Hydratase / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D007523:Isocitrates / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000032:analysis
chemical_list: D001426:Bacterial Proteins; D007523:Isocitrates; C034219:isocitric acid; D001616:beta-Galactosidase; D000154:Aconitate Hydratase