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

• Type of Material: Article
• Authors: Thomas McCorvie, Queens University Belfast; Ying Liu, Emory University; Andrew Frazer, Queens University Belfast; Tyler J. Gleason, Emory University; Judith Fridovich-Keil, Emory University; David J. Timson, Queens University Belfast
• Language: English
• Date: 2012-10-01
• Publisher: Elsevier: 12 months
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2012 Elsevier B.V.
• License: Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1016/j.bbadis.2012.05.007
• Title of Journal or Parent Work: Biochimica et Biophysica Acta Molecular and Cell Biology of Lipids
• ISSN: 1388-1981
• Volume: 1822
• Issue: 10
• Start Page: 1516
• End Page: 1526
• Grant/Funding Information: 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).
• Keywords: Cell Biology; UDP-galactose 4 '-epimerase; GALE; URIDINE-DIPHOSPHATE GALACTOSE-4-EPIMERASE; Biophysics; PHARMACOLOGICAL CHAPERONES; ESCHERICHIA-COLI; MOLECULAR-BIOLOGY; FUNCTIONAL-CHARACTERIZATION; Biochemistry & Molecular Biology; CRYSTAL-STRUCTURE; HUMAN UDP-GALACTOSE-4-EPIMERASE; AEROMONAS-HYDROPHILA; Yeast model; Life Sciences & Biomedicine; Type III galactosemia; LELOIR PATHWAY; Science & Technology; Differential scanning fluorimetry; EPIMERASE-DEFICIENCY GALACTOSEMIA; Disease-associated mutation
• Research Categories: Chemistry, Biochemistry; Biology, Cell; Biology, Genetics

Abstract

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.