title: Development of a colorimetric assay and kinetic analysis for Mycobacterium tuberculosis D-glucose-1-phosphate thymidylyltransferase.
abstract: dTDP-L-rhamnose as a sugar donor provides L-rhamnosyl residue in the synthesis of disaccharide linker (D-N-acetylglucosamine-L-rhamnose), the key structure of the Mycobacterium tuberculosis cell wall. Four enzymes are involved in the formation of dTDP-L-rhamnose and D-glucose-1-phosphate thymidylyltransferase (RmlA) catalyzes the first step of D-glucose-1-phosphate and dTTP to dTDP-D-glucose and PPi. The previous studies on RmlA essentiality proved RmlA as a potential target for antituberculosis drugs. However, there has not been a suitable assay for RmlA to screen inhibitors currently. In this study, the authors reported a microtiter plate-based colorimetric assay for RmlA enzyme activity. Using this assay, the kinetic properties of M. tuberculosis RmlA including initial velocity, optimal temperature, optimal pH, the effect of Mg(2+), and kinetic parameters were determined. The establishment of the accurate and rapid colorimetric assay and kinetic analysis of M. tuberculosis RmlA will facilitate high-throughput screening of RmlA inhibitors.
authors.affiliation: Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian, China.
authors.forename: Shanshan
authors.initials: S
authors.lastname: Sha
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhou
authors.forename: Yi
authors.initials: Y
authors.lastname: Xin
authors.forename: Yufang
authors.initials: Y
authors.lastname: Ma
mesh_terms: D000995:Antitubercular Agents; D002473:Cell Wall / Q000737:chemistry; D003124:Colorimetry / Q000379:methods*; D055808:Drug Discovery; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D009713:Nucleotidyltransferases / Q000032:analysis* / Q000037:antagonists & inhibitors; D014376:Tuberculosis / Q000188:drug therapy
chemical_list: D000995:Antitubercular Agents; D009713:Nucleotidyltransferases; C020587:glucose-1-phosphate thymidylyltransferase