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