Determinants of Function and Substrate Specificity in Human UDP-galactose 4′-Epimerase

Abstract

UDP-galactose 4-epimerase (GALE) interconvertsUDP-galactose and UDP-glucose in the final step of the Leloir pathway. Unlike the Escherichia coli enzyme, human GALE (hGALE) also efficiently interconverts a larger pair of substrates: UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine. The basis of this differential substrate specificity has remained obscure. Recently, however, x-ray crystallographic data have both predicted essential active site residues and suggested that differential active site cleft volume may be a key factor in determining GALE substrate selectivity. We report here a direct test of this hypothesis. In brief, we have created four substituted alleles: S132A, Y157F, S132A/Y157F, and C307Y-hGALE. While the first three substitutions were predicted to disrupt catalytic activity, the fourth was predicted to reduce active site cleft volume, thereby limiting entry or rotation of the larger but not the smaller substrate. All four alleles were expressed in a null-background strain of Saccharomyces cerevisiae and characterized in terms of activity with regard to both UDP-galactose and UDP-N-acetylgalactosamine. The S132A/Y157F and C307Y-hGALE proteins were also overexpressed in Pichia pastoris and purified for analysis. In all forms tested, the Y157F, S132A, and Y157F/S132A-hGALE proteins each demonstrated a complete loss of activity with respect to both substrates. In contrast, the C307Y-hGALE demonstrated normal activity with respect to UDP-galactose but complete loss of activity with respect to UDP-N-acetylgalactosamine. Together, these results serve to validate the wild-type hGALE crystal structure and fully support the hypothesis that residue 307 acts as a gatekeeper mediating substrate access to the hGALE active site.

Mutational Analysis of the Medicago Glycosyltransferase UGT71G1 Reveals Residues That Control Regioselectivity for (Iso)flavonoid Glycosylation

“…Cys 307 in the active site of the epimerase acts as a gatekeeper for substrate access to the site. The C307Y mutant completely lost activity for UDP-N-acetylgalactosamine, the larger substrate, but retained normal activity for UDP-galactose (31).…”

“…This binding pocket volume change would allow the 3-hydroxyl to move closer to the C1 reaction center on the UDP-glucose and be more favorably placed for glycosylation. Active site cleft volume was previously reported to determine the substrate specificity of human UDP-galactose 4Ј-epimerase, which interconverts UDP-galactose and UDP-glucose, and UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine (31). Cys 307 in the active site of the epimerase acts as a gatekeeper for substrate access to the site.…”

Altered architecture of substrate binding region defines the unique specificity of UDP‐GalNAc 4‐epimerases

“…The importance of this variation has been reported previously by a Tyr299Cys mutation engineered in the Group 1 epimerase GalE that switched its specificity from the Group 1 to Group 2, 20 by a Ser306Tyr mutation engineered into the Group 2 epimerase from E. coli O86:B7 that switched its specificity from the Group 2 to the Group 1 21 and the corresponding Cys307Tyr mutation in HGal that switched its activity from the Group 2 to the Group 1. 20,22 Second, can a single point mutation of Ser306-Tyr also switch the substrate specificity from the Group 3 to the Group 1? Based on our model of substrate recognition, a single point mutation of Ser306Tyr can not be engineered in the Group 3 epimerases since it would place the Tyr residue in steric clashes in all of the allowed rotamer conformations.…”

The molecular basis of galactosemia — Past, present and future

“…The most common mutation associated with a severe form of the disease, which codes for p.V94M, was discovered in 1999 (Wohlers et al , 1999). In addition to its role in the Leloir pathway, human GALE also catalyses the interconversion of N-acetylgalactosamine and N-acetylglucoasamine (Piller et al , 1983;Schulz et al , 2004). This reaction is important in maintaining the pools of UDP-sugars used in the synthesis of glycoproteins and glycolipids and loss of this activity may explain the abnormal glycosylation patterns seen in some cell culture and animal models of type III galactosemia (Kingsley et al , 1986;Rosoff , 1995;BrokateLlanos et al , 2014).…”