title: Unraveling the B. pseudomallei Heptokinase WcbL: From Structure to Drug Discovery.
abstract: Gram-negative bacteria utilize heptoses as part of their repertoire of extracellular polysaccharide virulence determinants. Disruption of heptose biosynthesis offers an attractive target for novel antimicrobials. A critical step in the synthesis of heptoses is their 1-O phosphorylation, mediated by kinases such as HldE or WcbL. Here, we present the structure of WcbL from Burkholderia pseudomallei. We report that WcbL operates through a sequential ordered Bi-Bi mechanism, loading the heptose first and then ATP. We show that dimeric WcbL binds ATP anti-cooperatively in the absence of heptose, and cooperatively in its presence. Modeling of WcbL suggests that heptose binding causes an elegant switch in the hydrogen-bonding network, facilitating the binding of a second ATP molecule. Finally, we screened a library of drug-like fragments, identifying hits that potently inhibit WcbL. Our results provide a novel mechanism for control of substrate binding and emphasize WcbL as an attractive anti-microbial target for Gram-negative bacteria.
authors.affiliation: Department of Biosciences, University of Exeter, Henry Wellcome Building, Stocker Road, Exeter EX4 4QD, UK.
authors.forename: Mirella
authors.initials: M
authors.lastname: Vivoli
authors.affiliation: Department of Biosciences, University of Exeter, Henry Wellcome Building, Stocker Road, Exeter EX4 4QD, UK.
authors.forename: Michail N
authors.initials: MN
authors.lastname: Isupov
authors.affiliation: Department of Biosciences, University of Exeter, Henry Wellcome Building, Stocker Road, Exeter EX4 4QD, UK.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Nicholas
authors.affiliation: Department of Biosciences, University of Exeter, Henry Wellcome Building, Stocker Road, Exeter EX4 4QD, UK.
authors.forename: Andrew
authors.initials: A
authors.lastname: Hill
authors.affiliation: Defence Science and Technology Laboratory, Porton Down, Salisbury, Wiltshire SP4 0JQ, UK.
authors.forename: Andrew E
authors.initials: AE
authors.lastname: Scott
authors.affiliation: University of Natural Resources and Life Sciences-Vienna, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Paul
authors.initials: P
authors.lastname: Kosma
authors.affiliation: Defence Science and Technology Laboratory, Porton Down, Salisbury, Wiltshire SP4 0JQ, UK.
authors.forename: Joann L
authors.initials: JL
authors.lastname: Prior
authors.affiliation: Department of Biosciences, University of Exeter, Henry Wellcome Building, Stocker Road, Exeter EX4 4QD, UK. Electronic address: n.j.harmer@exeter.ac.uk.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Harmer
mesh_terms: D016957:Burkholderia pseudomallei / Q000201:enzymology*; D002152:Calorimetry, Differential Scanning; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery; D004353:Drug Evaluation, Preclinical; D006539:Heptoses / Q000737:chemistry; D008958:Models, Molecular; D010770:Phosphotransferases / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D006539:Heptoses; D054852:Small Molecule Libraries; D010770:Phosphotransferases