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

Thomas J. McCorvie, Ying Liu, Andrew Frazer, Tyler J. Gleason, Judith L. Fridovich-Keil, David J. Timson

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    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.

    Original languageEnglish
    Pages (from-to)1516-1526
    Number of pages11
    JournalBiochimica et Biophysica Acta - Molecular Basis of Disease
    Issue number10
    Publication statusPublished - 1 Oct 2012


    • Differential scanning fluorimetry
    • Disease-associated mutation
    • GALE
    • Type III galactosemia
    • UDP-galactose 4'-epimerase
    • Yeast model


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