UDP-galactose 4'-epimerase from the liver fluke, Fasciola hepatica: biochemical characterization of the enzyme and identification of inhibitors

Veronika L Zinsser, Steffen Lindert, Samantha Banford, Elizabeth M Hoey, Alan Trudgett, David J Timson

Research output: Contribution to journalArticle

3 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)463-72
Number of pages10
JournalParasitology
Volume142
Issue number3
Early online date15 Aug 2014
DOIs
Publication statusPublished - Mar 2015

Fingerprint

Uridine Diphosphate Galactose
liver flukes
Fasciola hepatica
Enzyme Inhibitors
Enzymes
enzymes
uridine diphosphate
orotic acid
Orotic Acid
fluorine
National Cancer Institute (U.S.)
Fluorine
active sites
galactose
Computer Simulation
NAD
Inhibitory Concentration 50
Libraries
Gel Chromatography
Tyrosine

Cite this

Zinsser, Veronika L ; Lindert, Steffen ; Banford, Samantha ; Hoey, Elizabeth M ; Trudgett, Alan ; Timson, David J. / UDP-galactose 4'-epimerase from the liver fluke, Fasciola hepatica: biochemical characterization of the enzyme and identification of inhibitors. In: Parasitology. 2015 ; Vol. 142, No. 3. pp. 463-72.
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UDP-galactose 4'-epimerase from the liver fluke, Fasciola hepatica: biochemical characterization of the enzyme and identification of inhibitors. / Zinsser, Veronika L; Lindert, Steffen; Banford, Samantha; Hoey, Elizabeth M; Trudgett, Alan; Timson, David J.

In: Parasitology, Vol. 142, No. 3, 03.2015, p. 463-72.

Research output: Contribution to journalArticle

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AU - Zinsser, Veronika L

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AB - 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.

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