Microbiology Division, Central Drug Research Institute, CSIR, Lucknow 226001, India.
Department of Biochemistry, University of Lucknow, Lucknow 226001, India.
Microbiology (Reading). 2011 Jan;157(Pt 1):38-46. doi: 10.1099/mic.0.042358-0. Epub 2010 Sep 23.
Dihydroxyacid dehydratase (DHAD), a key enzyme involved in branched-chain amino acid (BCAA) biosynthesis, catalyses the synthesis of 2-ketoacids from dihydroxyacids. In Mycobacterium tuberculosis, DHAD is encoded by gene Rv0189c, and it shares 40% amino acid sequence identity and conserved motifs with DHAD of Escherichia coli encoded by ilvD. In this study, Rv0189c was overexpressed in E. coli and the resultant protein was characterized as a homodimer (~155 kDa). Functional characterization of Rv0189c was established by biochemical testing and by genetic complementation of an intron-disrupted ilvD-auxotrophic mutant of E. coli to prototrophy. Growth of M. tuberculosis, E. coli BL21(DE3) and recombinant E. coli BL21(DE3) ΔilvD carrying Rv0189c was inhibited by transient nitric oxide (NO) exposure in minimal medium but growth was restored if the medium was supplemented with BCAA (isoleucine, leucine and valine). This suggested that inactivation of Rv0189c by NO probably inhibited bacterial growth. The role of Rv0189c in M. tuberculosis was elucidated by antisense and sense RNA constructs. Growth of M. tuberculosis transformed with a plasmid encoding antisense mRNA was markedly poor in the lungs of infected mice and in Middlebrook 7H9 broth compared to that of sense and vector-alone transformants, but growth was normal when the medium was supplemented with BCAA. Upregulation of Rv0189c was observed during the early exponential phase of growth, under acid stress and ex vivo, suggesting that Rv0189c has a role in the survival of M. tuberculosis during normal and stress conditions. It may be concluded that the DHAD encoded by Rv0189c is essential for the survival of M. tuberculosis and could be a potential drug/vaccine target, as it is absent in mammals.
二羟酸脱水酶（DHAD）是支链氨基酸（BCAA）生物合成中的关键酶，催化二羟酸合成 2-酮酸。在结核分枝杆菌中，DHAD 由基因 Rv0189c 编码，它与大肠杆菌编码的 ilvD 的 DHAD 具有 40%的氨基酸序列同一性和保守基序。在本研究中，Rv0189c 在大肠杆菌中过表达，所得蛋白为同源二聚体（~155 kDa）。通过生化测试和对大肠杆菌 ilvD 内含子缺失的营养缺陷型突变体进行遗传互补，证明了 Rv0189c 的功能。在最小培养基中，短暂的一氧化氮（NO）暴露会抑制结核分枝杆菌、大肠杆菌 BL21(DE3) 和携带 Rv0189c 的重组大肠杆菌 BL21(DE3)ΔilvD 的生长，但如果培养基中补充了 BCAA（异亮氨酸、亮氨酸和缬氨酸），生长就会恢复。这表明 NO 可能通过失活 Rv0189c 抑制了细菌的生长。通过反义和正义 RNA 构建物阐明了 Rv0189c 在结核分枝杆菌中的作用。与携带编码正义 mRNA 的质粒转化的结核分枝杆菌相比，感染小鼠肺部和 Middlebrook 7H9 肉汤中的生长明显较差，而在补充 BCAA 的培养基中生长正常。在生长的早期指数期、酸胁迫和体外观察到 Rv0189c 的上调，表明 Rv0189c 在结核分枝杆菌正常和应激条件下的生存中起作用。可以得出结论，Rv0189c 编码的 DHAD 对结核分枝杆菌的生存是必不可少的，并且可能是一种潜在的药物/疫苗靶标，因为它在哺乳动物中不存在。
