title: UDP-galactose 4'-epimerase from the liver fluke, Fasciola hepatica: biochemical characterization of the enzyme and identification of inhibitors.
abstract: Leloir pathway enzyme uridine diphosphate (UDP)-galactose 4'-epimerase from the common liver fluke Fasciola hepatica (FhGALE) was identified and characterized. The enzyme can be expressed in, and purified from, Escherichia coli. The recombinant enzyme is active: the K(m) (470 μM) is higher than the corresponding human enzyme (HsGALE), whereas the k(cat) (2.3 s(-1)) is substantially lower. FhGALE binds NAD(+) and has shown to be dimeric by analytical gel filtration. Like the human and yeast GALEs, FhGALE is stabilized by the substrate UDP-galactose. Molecular modelling predicted that FhGALE adopts a similar overall fold to HsGALE and that tyrosine 155 is likely to be the catalytically critical residue in the active site. In silico screening of the National Cancer Institute Developmental Therapeutics Program library identified 40 potential inhibitors of FhGALE which were tested in vitro. Of these, 6 showed concentration-dependent inhibition of FhGALE, some with nanomolar IC50 values. Two inhibitors (5-fluoroorotate and N-[(benzyloxy)carbonyl]leucyltryptophan) demonstrated selectivity for FhGALE over HsGALE. These compounds also thermally destabilized FhGALE in a concentration-dependent manner. Interestingly, the selectivity of 5-fluoroorotate was not shown by orotic acid, which differs in structure by 1 fluorine atom. These results demonstrate that, despite the structural and biochemical similarities of FhGALE and HsGALE, it is possible to discover compounds which preferentially inhibit FhGALE.
authors.affiliation: School of Biological Sciences, Queen's University Belfast, Medical Biology Centre,97 Lisburn Road, Belfast BT9 7BL,UK.
authors.forename: Veronika L
authors.initials: VL
authors.lastname: Zinsser
authors.affiliation: Department of Pharmacology,Center for Theoretical Biological Physics, University of California San Diego,La Jolla, CA 92093,USA.
authors.forename: Steffen
authors.initials: S
authors.lastname: Lindert
authors.affiliation: School of Biological Sciences, Queen's University Belfast, Medical Biology Centre,97 Lisburn Road, Belfast BT9 7BL,UK.
authors.forename: Samantha
authors.initials: S
authors.lastname: Banford
authors.affiliation: School of Biological Sciences, Queen's University Belfast, Medical Biology Centre,97 Lisburn Road, Belfast BT9 7BL,UK.
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Hoey
authors.affiliation: School of Biological Sciences, Queen's University Belfast, Medical Biology Centre,97 Lisburn Road, Belfast BT9 7BL,UK.
authors.forename: Alan
authors.initials: A
authors.lastname: Trudgett
authors.affiliation: School of Biological Sciences, Queen's University Belfast, Medical Biology Centre,97 Lisburn Road, Belfast BT9 7BL,UK.
authors.forename: David J
authors.initials: DJ
authors.lastname: Timson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005210:Fasciola hepatica / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D020128:Inhibitory Concentration 50; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D014534:UDPglucose 4-Epimerase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics
chemical_list: D004791:Enzyme Inhibitors; D014534:UDPglucose 4-Epimerase
keywords: N-[(benzyloxy)carbonyl]leucyltryptophan