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Author Notes:

David J. Timson School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast, BT9 7BL, UK; Tel.: + 44 28 9097 5875; fax: + 44 28 9097 5877; Email: d.timson@qub.ac.uk.

TJM thanks the Department of Employment and Learning, Northern Ireland for a PhD studentship.

We wish to thank Prof Aaron Maule (Queen's University, Belfast) for use of a thermal cycler for the thermal scanning fluorimetry assay and Dr. Kostya Panov (Queen's University, Belfast) for suggesting this assay.

The authors have no conflicts of interest to declare.


Research Funding:

Work conducted in the Fridovich-Keil lab was supported, in part, by funds from the National Institutes of Health (USA) grant R01 DK059904 (to JLFK).


  • Science & Technology
  • Life Sciences & Biomedicine
  • Biochemistry & Molecular Biology
  • Biophysics
  • Cell Biology
  • Type III galactosemia
  • Yeast model
  • GALE
  • Disease-associated mutation
  • UDP-galactose 4 '-epimerase
  • Differential scanning fluorimetry

Altered cofactor binding affects stability and activity of human UDP-galactose 4′-epimerase: implications for type III galactosemia


Journal Title:

Biochimica et Biophysica Acta Molecular and Cell Biology of Lipids


Volume 1822, Number 10


, Pages 1516-1526

Type of Work:

Article | Post-print: After Peer Review


Deficiency of UDP-galactose 4'-epimerase is implicated in type III galactosemia. Two variants, p.K161N-hGALE and p.D175N-hGALE, have been previously found in combination with other alleles in patients with a mild form of the disease. Both variants were studied in vivo and in vitro and showed different levels of impairment. p.K161N-hGALE was severely impaired with substantially reduced enzymatic activity, increased thermal stability, reduced cofactor binding and no ability to rescue the galactose-sensitivity of gal10-null yeast. Interestingly p.K161N-hGALE showed less impairment of activity with UDP-N-acetylgalactosamine in comparison to UDP-galactose. Differential scanning fluorimetry revealed that p.K161N-hGALE was more stable than the wild-type protein and only changed stability in the presence of UDP-N-acetylglucosamine and NAD + . p.D175N-hGALE essentially rescued the galactose-sensitivity of gal10-null yeast, was less stable than the wild-type protein but showed increased stability in the presence of substrates and cofactor. We postulate that p.K161N-hGALE causes its effects by abolishing an important interaction between the protein and the cofactor, whereas p.D175N-hGALE is predicted to remove a stabilizing salt bridge between the ends of two α-helices that contain residues that interact with NAD + . These results suggest that the cofactor binding is dynamic and that its loss results in significant structural changes that may be important in disease causation.

Copyright information:

© 2012 Elsevier B.V.

This is an Open Access work distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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