A 96-well microtiter plate assay for high-throughput screening of Mycobacterium tuberculosis dTDP-d-glucose 4,6-dehydratase inhibitors

Analytical Biochemistry
Xiaoxia ShiYufang Ma

Abstract

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.

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Citations

Nov 12, 2016·Plant Physiology and Biochemistry : PPB·Sen YinJian-Qiang Kong
Nov 17, 2016·Chemical Communications : Chem Comm·Siqiang LiZhongwu Guo
Dec 3, 2016·The Review of Scientific Instruments·Enoch Ming Wei NgTuck Wah Ng
Mar 21, 2020·Nature Communications·Qinqin ZhaoZixin Deng

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