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 inability 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.

Misfolding of galactose 1-phosphate uridylyltransferase can result in type I galactosemia

“…Differential scanning fluorimetry was carried out essentially as previously described [39;40]. Protein samples were diluted in 10 mM HEPES, pH 8.8 to a final concentration of 5 μM and any ligands used were added at a final concentration of 1 mM.…”

Comparison of dynamics of wildtype and V94M human UDP-galactose 4-epimerase—A computational perspective on severe epimerase-deficiency galactosemia

“…K161N is also very severely impaired kinetically, with the likely loss of the NAD cofactor. Interestingly, this mutant is thermally more stable than wildtype (18). …”

“…In addition, aggregation of HsGALE has been observed in cells expressing disease-associated variants (22). As yet, there are no small molecule therapies for any form of galactosemia, although it has been suggested that a “small molecule chaperone” approach may be viable since many of the mutations result in decreased protein stability (1, 15, 17, 18, 23-25). In order to implement such therapies, however, it is important to understand more about not just the structures of the proteins and the disease-associated variants but also the flexibility and dynamics of these structures.…”

UDP-galactose 4′-epimerase from the liver fluke,Fasciola hepatica: biochemical characterization of the enzyme and identification of inhibitors

“…Irradiation of the protein at 280 nm results in a large tryptophan emission peak at ~330 nm. However, some of the emission transfers to the NAD + which emits at ~420 nm (McCorvie et al 2012). A similar effect was observed with FhGALE (Figure 3a).…”

“…GALE has been shown to be essential for normal development in Drosophila melanogaster (Daenzer et al 2012; Sanders et al 2010). Although the molecular pathology of type III galactosemia is not completely elucidated, the fundamental molecular cause has been shown to be protein misfolding which results in reduced enzymatic activity, reduced cofactor binding or an increased propensity to aggregate (Bang et al 2009; Chhay et al 2008; McCorvie et al 2011; McCorvie et al 2012; Timson 2005; Wohlers and Fridovich-Keil 2000). A reduction in GALE activity results in a build-up of galactose 1-phosphate, which is believed to be toxic in high concentrations (Mayes et al 1970; Tsakiris et al 2005).…”