title: Altered cofactor binding affects stability and activity of human UDP-galactose 4'-epimerase: implications for type III galactosemia.
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.
authors.affiliation: School of Biological Sciences, Queen's University, Belfast, BT9 7BL, UK.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: McCorvie
authors.forename: Ying
authors.initials: Y
authors.lastname: Liu
authors.forename: Andrew
authors.initials: A
authors.lastname: Frazer
authors.forename: Tyler J
authors.initials: TJ
authors.lastname: Gleason
authors.forename: Judith L
authors.initials: JL
authors.lastname: Fridovich-Keil
authors.forename: David J
authors.initials: DJ
authors.lastname: Timson
mesh_terms: D000483:Alleles; D003067:Coenzymes / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D005690:Galactose / Q000235:genetics / Q000378:metabolism; D005693:Galactosemias / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009243:NAD / Q000235:genetics / Q000378:metabolism; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics*; D011489:Protein Denaturation; D055503:Protein Multimerization; D017433:Protein Structure, Secondary / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D014534:UDPglucose 4-Epimerase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000235:genetics / Q000378:metabolism; D015003:Yeasts / Q000235:genetics / Q000378:metabolism
chemical_list: D003067:Coenzymes; D005656:Fungal Proteins; D011994:Recombinant Proteins; D009243:NAD; D014537:Uridine Diphosphate N-Acetylglucosamine; D010447:Peptide Hydrolases; D014534:UDPglucose 4-Epimerase; D005690:Galactose