title: A 96-well microtiter plate assay for high-throughput screening of Mycobacterium tuberculosis dTDP-d-glucose 4,6-dehydratase inhibitors.
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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044, China.
authors.forename: Xiaoxia
authors.initials: X
authors.lastname: Shi
authors.affiliation: Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044, China.
authors.forename: Shanshan
authors.initials: S
authors.lastname: Sha
authors.affiliation: Department of Biotechnology, Dalian Medical University, Dalian 116044, China.
authors.forename: Likun
authors.initials: L
authors.lastname: Liu
authors.affiliation: Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044, China. Electronic address: yufang_ma@hotmail.com.
authors.forename: Yufang
authors.initials: Y
authors.lastname: Ma
mesh_terms: D000995:Antitubercular Agents / Q000737:chemistry / Q000494:pharmacology*; D001681:Biological Assay; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D005947:Glucose / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D057166:High-Throughput Screening Assays; D006836:Hydro-Lyases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology* / Q000302:isolation & purification; D013942:Thymine Nucleotides / Q000737:chemistry / Q000494:pharmacology
chemical_list: D000995:Antitubercular Agents; D004791:Enzyme Inhibitors; D013942:Thymine Nucleotides; C103522:deoxythymidine diphosphate-glucose; D006836:Hydro-Lyases; C022771:dTDPglucose 4,6-dehydratase; D005947:Glucose
keywords: High-throughput screening; Inhibitor; Kinetic properties; Microtiter plate assay; Mycobacterium tuberculosis; RmlB