| Solution Information | help | |
| Enzyme: | dTDP-4-dehydrorhamnose 3,5-epimerase | |
| inhibitor: | BDBM51582 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | MATERIALS The purified rhamnosyl biosynthetic enzymes RmlB, RmlC and RmlD, cloned and expressed in E. coli, were provided by Michael McNeil of Colorado State University. MOPS, TritonX 100 and TDP-Glc were purchased from Sigma. MgCl2 and glycerol were purchased from Fisher. NADPH was purchased from Roche. The RmlC enzyme substrate, dTDP-4-keto-6-deoxy-D-xylo-hexulose (dTDP-KDX), was synthesized enzymatically by converting d-TDP-glucose (2.5 mg) to dTDP-KDX using RmlB (2.4 ug), in 50 mM MOPS buffer, pH 7.4, at 37 deg C for 1 hr. Aliquots of dTDP-KDX were stored at -80 deg C. Each aliquot was subjected to freeze thaw no more than three times. Assay plate---Black 384-well plate (Corning 3676) Serial dilution compound plate---Polypropylene 384-well V-bottom plate (Greiner 781280) ASSAY The screen for inhibitors of the Mycobacterium tuberculosis cell wall enzymes, RmlC and RmlD, reported here is based on the decrease in fluorescence observed upon the oxidation of NADPH to NADP. Chang | |
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