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

• 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
• Journal Title: Biochimica et Biophysica Acta Molecular and Cell Biology of Lipids
• Volume: Volume 1822, Number 10
• Publisher: Elsevier: 12 months | 2012-10-01, Pages 1516-1526
• Type of Work: Article | Post-print: After Peer Review
• Publisher DOI: http://doi.org/10.1016/j.bbadis.2012.05.007
• Subjects: Biology, Genetics; Chemistry, Biochemistry; Biology, Cell
• Keywords: 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; EPIMERASE-DEFICIENCY GALACTOSEMIA; URIDINE-DIPHOSPHATE GALACTOSE-4-EPIMERASE; HUMAN UDP-GALACTOSE-4-EPIMERASE; FUNCTIONAL-CHARACTERIZATION; PHARMACOLOGICAL CHAPERONES; AEROMONAS-HYDROPHILA; MOLECULAR-BIOLOGY; CRYSTAL-STRUCTURE; ESCHERICHIA-COLI; LELOIR PATHWAY

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.