Shi Xiaoxia, Sha Shanshan, Liu Likun, Li Xin, Ma Yufang
Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044, China.
Department of Biotechnology, Dalian Medical University, Dalian 116044, China.
Anal Biochem. 2016 Apr 1;498:53-8. doi: 10.1016/j.ab.2016.01.004. Epub 2016 Jan 15.
Mycobacterium tuberculosis dTDP-d-glucose 4,6-dehydratase (RmlB) is the second enzyme for the biosynthesis of dTDP-l-rhamnose, which is a sugar donor to the synthesis of the cell wall linker, d-N-acetylglucosamine-l-rhamnose. RmlB is essential to mycobacterial growth and is not found in humans; therefore, it is a potential target for developing new anti-tuberculosis drugs. So far, there has been no suitable method for high-throughput screening of RmlB inhibitors. Here, the recombinant M. tuberculosis RmlB was purified and an absorbance-based microtiter plate assay was developed for RmlB activity. It could be used for high-throughput screening of RmlB inhibitors. The kinetic properties of M. tuberculosis RmlB, including optimal pH, optimal temperature, the effect of metal ions, and the kinetic parameters, were determined with this assay. The inhibitory effects of dTTP and dTDP on M. tuberculosis RmlB were also studied with the assay.
结核分枝杆菌dTDP - D - 葡萄糖4,6 - 脱水酶（RmlB）是dTDP - L - 鼠李糖生物合成途径中的第二个酶，dTDP - L - 鼠李糖是细胞壁连接物d - N - 乙酰葡糖胺 - L - 鼠李糖合成的糖供体。RmlB对分枝杆菌生长至关重要，且在人体内不存在；因此，它是开发新型抗结核药物的潜在靶点。到目前为止，还没有适合高通量筛选RmlB抑制剂的方法。在此，我们纯化了重组结核分枝杆菌RmlB，并开发了一种基于吸光度的微孔板测定法来检测RmlB活性。该方法可用于高通量筛选RmlB抑制剂。利用该测定法确定了结核分枝杆菌RmlB的动力学性质，包括最适pH、最适温度、金属离子的影响以及动力学参数。还用该测定法研究了dTTP和dTDP对结核分枝杆菌RmlB的抑制作用。
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