Movahedzadeh Farahnaz, Smith Debbie A, Norman Richard A, Dinadayala Premkumar, Murray-Rust Judith, Russell David G, Kendall Sharon L, Rison Stuart C G, McAlister Mark S B, Bancroft Gregory J, McDonald Neil Q, Daffe Mamadou, Av-Gay Yossef, Stoker Neil G
Department of Pathology and Infectious Diseases, Royal Veterinary College, London, UK.
Mol Microbiol. 2004 Feb;51(4):1003-14. doi: 10.1046/j.1365-2958.2003.03900.x.
Inositol is utilized by Mycobacterium tuberculosis in the production of its major thiol and of essential cell wall lipoglycans. We have constructed a mutant lacking the gene encoding inositol-1-phosphate synthase (ino1), which catalyses the first committed step in inositol synthesis. This mutant is only viable in the presence of extremely high levels of inositol. Mutant bacteria cultured in inositol-free medium for four weeks showed a reduction in levels of mycothiol, but phosphatidylinositol mannoside, lipomannan and lipoarabinomannan levels were not altered. The ino1 mutant was attenuated in resting macrophages and in SCID mice. We used site-directed mutagenesis to alter four putative active site residues; all four alterations resulted in a loss of activity, and we demonstrated that a D310N mutation caused loss of the active site Zn2+ ion and a conformational change in the NAD+ cofactor.
肌醇被结核分枝杆菌用于合成其主要硫醇和必需的细胞壁脂聚糖。我们构建了一个缺失编码肌醇 -1-磷酸合酶（ino1）基因的突变体，该酶催化肌醇合成的第一步关键反应。此突变体仅在存在极高水平肌醇的情况下才能存活。在无肌醇培养基中培养四周的突变细菌显示，巯基乙醇水平降低，但磷脂酰肌醇甘露糖苷、脂甘露聚糖和脂阿拉伯甘露聚糖水平未改变。ino1突变体在静息巨噬细胞和SCID小鼠中减毒。我们使用定点诱变来改变四个假定的活性位点残基；所有四个改变均导致活性丧失，并且我们证明D310N突变导致活性位点Zn2+离子丢失以及NAD+辅因子的构象变化。
