title: Biosynthesis of a water-soluble lipid I analogue and a convenient assay for translocase I.
abstract: Translocase I (MraY/MurX) is an essential enzyme in growth of the vast majority of bacteria that catalyzes the transformation from UDP-MurNAc-pentapeptide (Park's nucleotide) to prenyl-MurNAc-pentapeptide (lipid I), the first membrane-anchored peptidoglycan precursor. MurX has received considerable attention in the development of new tuberculosis (TB) drugs due to the fact that the MurX inhibitors kill exponentially growing Mycobacterium tuberculosis (Mtb) much faster than clinically used TB drugs. Lipid I isolated from Mtb contains the C50-prenyl unit that shows very poor water solubility; thus, this chemical characteristic of lipid I renders MurX enzyme assays impractical for screening and lacks reproducibility of the enzyme assays. We have established a scalable chemical synthesis of Park's nucleotide-N(ε)-dansylthiourea 2 that can be used as a MurX enzymatic substrate to form lipid I analogues. In our investigation of the minimum structure requirement of the prenyl phosphate in the MraY/MurX-catalyzed lipid I analogue synthesis with 2, we found that neryl phosphate (C10 phosphate) can be recognized by MraY/MurX to generate the water-soluble lipid I analogue in quantitative yield under the optimized conditions. Here, we report a rapid and robust analytical method for quantifying MraY/MurX inhibitory activity of library molecules.
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, 881 Madison Avenue, Memphis, TN 38163-0001, United States.
authors.forename: Shajila
authors.initials: S
authors.lastname: Siricilla
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, 881 Madison Avenue, Memphis, TN 38163-0001, United States.
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Mitachi
authors.affiliation: Department of Lipid Biochemistry, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warszawa, Poland.
authors.forename: Karolina
authors.initials: K
authors.lastname: Skorupinska-Tudek
authors.affiliation: Department of Lipid Biochemistry, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warszawa, Poland.
authors.forename: Ewa
authors.initials: E
authors.lastname: Swiezewska
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, 881 Madison Avenue, Memphis, TN 38163-0001, United States.
authors.forename: Michio
authors.initials: M
authors.lastname: Kurosu
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D008826:Microbial Sensitivity Tests; D009005:Monosaccharides / Q000096:biosynthesis* / Q000737:chemistry*; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology; D009842:Oligopeptides / Q000096:biosynthesis* / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology; D012995:Solubility; D014166:Transferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017855:Transferases (Other Substituted Phosphate Groups); D014529:Uridine / Q000737:chemistry; D014867:Water / Q000737:chemistry
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D009005:Monosaccharides; D009842:Oligopeptides; D010455:Peptides; C438940:lipid I; D014867:Water; D014166:Transferases; D017855:Transferases (Other Substituted Phosphate Groups); C079803:mraY protein, Bacteria; D014529:Uridine
keywords: MraY; MurX; MurX inhibitors; Mycobacterium tuberculosis; Translocase I; Water-soluble lipid I