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

Published online by Cambridge University Press:  15 August 2014

VERONIKA L. ZINSSER ,
STEFFEN LINDERT ,
SAMANTHA BANFORD ,
ELIZABETH M. HOEY ,
ALAN TRUDGETT  and
DAVID J. TIMSON Open the ORCID record for DAVID J. TIMSON [Opens in a new window]
VERONIKA L. ZINSSER
Affiliation:
School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UK
STEFFEN LINDERT
Affiliation:
Department of Pharmacology, Center for Theoretical Biological Physics, University of California San Diego, La Jolla, CA 92093, USA
SAMANTHA BANFORD
Affiliation:
School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UK
ELIZABETH M. HOEY
Affiliation:
School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UK
ALAN TRUDGETT
Affiliation:
School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UK Institute for Global Food Security, Queen's University Belfast, 18-30 Malone Road, Belfast BT9 5BN, UK
DAVID J. TIMSON*
Affiliation:
School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UK Institute for Global Food Security, Queen's University Belfast, 18-30 Malone Road, Belfast BT9 5BN, UK
*
*Corresponding author. School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UK. E-mail: d.timson@qub.ac.uk
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Summary

The 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 Km (470 μm) is higher than the corresponding human enzyme (HsGALE), whereas the kcat (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.

Keywords

neglected tropical diseaseLeloir pathwayGALEUDP-glucose 4′epimerase5-fluoroorotateN-[(benzyloxy)carbonyl]leucyltryptophan
Type
Research Article
Information
Parasitology , Volume 142 , Issue 3 , March 2015 , pp. 463 - 472
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Copyright
Copyright © Cambridge University Press 2014 

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Zinsser Supplementary Material

Figure S3

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