Human UDP-galactose 4-epimerase. Accommodation of UDP-N-acetylglucosamine within the active site.

UDP-galactose 4-epimerase catalyzes the interconversion of UDP-galactose and UDP-glucose during normal galactose metabolism. One of the key structural features in the proposed reaction mechanism for the enzyme is the rotation of a 4'-ketopyranose intermediate within the active site pocket. Recently, the three-dimensional structure of the human enzyme with bound NADH and UDP-glucose was determined. Unlike that observed for the protein isolated from Escherichia coli, the human enzyme can also turn over UDP-GlcNAc to UDP-GalNAc and vice versa. Here we describe the three-dimensional structure of human epimerase complexed with NADH and UDP-GlcNAc. To accommodate the additional N-acetyl group at the C2 position of the sugar, the side chain of Asn-207 rotates toward the interior of the protein and interacts with Glu-199. Strikingly, in the human enzyme, the structural equivalent of Tyr-299 in the E. coli protein is replaced with a cysteine residue (Cys-307) and the active site volume for the human protein is calculated to be approximately 15% larger than that observed for the bacterial epimerase. This combination of a larger active site cavity and amino acid residue replacement most likely accounts for the inability of the E. coli enzyme to interconvert UDP-GlcNAc and UDP-GalNAc.

Molecular basis for severe epimerase deficiency galactosemia. X-ray structure of the human V94m-substituted UDP-galactose 4-epimerase.

Galactosemia is an inherited disorder characterized by an inability to metabolize galactose. Although classical galactosemia results from impairment of the second enzyme of the Leloir pathway, namely galactose-1-phosphate uridylyltransferase, alternate forms of the disorder can occur due to either galactokinase or UDP-galactose 4-epimerase deficiencies. One of the more severe cases of epimerase deficiency galactosemia arises from an amino acid substitution at position 94. It has been previously demonstrated that the V94M protein is impaired relative to the wild-type enzyme predominantly at the level of V(max) rather than K(m). To address the molecular consequences the mutation imparts on the three-dimensional architecture of the enzyme, we have solved the structures of the V94M-substituted human epimerase complexed with NADH and UDP-glucose, UDP-galactose, UDP-GlcNAc, or UDP-GalNAc. In the wild-type enzyme, the hydrophobic side chain of Val(94) packs near the aromatic group of the catalytic Tyr(157) and serves as a molecular "fence" to limit the rotation of the glycosyl portions of the UDP-sugar substrates within the active site. The net effect of the V94M substitution is an opening up of the Ala(93) to Glu(96) surface loop, which allows free rotation of the sugars into nonproductive binding modes.

Studies of the V94M-substituted human UDPgalactose-4-epimerase enzyme associated with generalized epimerase-deficiency galactosaemia.

Impairment of the human enzyme UDPgalactose 4-epimerase (hGALE) results in epimerase-deficiency galactosaemia, an inborn error of metabolism with variable biochemical presentation and clinical outcomes reported to range from benign to severe. Molecular studies of the hGALE loci from patients with epimerase deficiency reveal significant allelic heterogeneity, raising the possibility that variable genotypes may constitute at least one factor contributing to the biochemical and clinical heterogeneity observed. Previously we have identified a single substitution mutation, V94M, present in the homozygous state in all patients genotyped with the severe, generalized form of epimerase-deficiency galactosaemia. We report here further studies of the V94M-hGALE enzyme, overexpressed and purified from a null-background yeast expression system. Our results demonstrate that the mutant protein is impaired relative to the wild-type enzyme predominantly at the level of Vmax rather than of Km. Studies using UDP-N-acetylgalactosamine as a competitor of UDPgalactose further demonstrate that the Km values for these two substrates vary by less than a factor of 3 for both the wild-type and mutant proteins. Finally, we have explored the impact of the V94M substitution on susceptibility of yeast expressing human GALE to galactose toxicity, including changes in the levels of galactose 1-phosphate (gal-1-P) accumulated in these cells at different times following exposure to galactose. We have observed an inverse correlation between the level of GALE activity expressed in a given culture and the degree of galactose toxicity observed. We have further observed an inverse correlation between the level of GALE activity expressed in a culture and the concentration of gal-1-P accumulated in the cells. These data support the hypothesis that elevated levels of gal-1-P may underlie the observed toxicity. They further raise the intriguing possibility that yeast may provide a valuable model not only for assessing the impact of given patient mutations on hGALE function, but also for exploring the metabolic imbalance resulting from impaired activity of GALE in living cells.

Crystallographic evidence for Tyr 157 functioning as the active site base in human UDP-galactose 4-epimerase.

UDP-galactose 4-epimerase catalyzes the interconversion of UDP-glucose and UDP-galactose during normal galactose metabolism. In humans, deficiencies in this enzyme lead to the complex disorder referred to as epimerase-deficiency galactosemia. Here, we describe the high-resolution X-ray crystallographic structures of human epimerase in the resting state (i.e., with bound NAD(+)) and in a ternary complex with bound NADH and UDP-glucose. Those amino acid side chains responsible for anchoring the NAD(+) to the protein include Asp 33, Asn 37, Asp 66, Tyr 157, and Lys 161. The glucosyl group of the substrate is bound to the protein via the side-chain carboxamide groups of Asn 187 and Asn 207. Additionally, O(gamma) of Ser 132 and O(eta) of Tyr 157 lie within 2.4 and 3.1 A, respectively, of the 4'-hydroxyl group of the sugar. Comparison of the polypeptide chains for the resting enzyme and for the protein with bound NADH and UDP-glucose demonstrates that the major conformational changes which occur upon substrate binding are limited primarily to the regions defined by Glu 199 to Asp 240 and Gly 274 to Tyr 308. Additionally, this investigation reveals for the first time that a conserved tyrosine, namely Tyr 157, is in the proper position to interact directly with the 4'-hydroxyl group of the sugar substrate and to thus serve as the active-site base. A low barrier hydrogen bond between the 4'-hydroxyl group of the sugar and O(gamma) of Ser 132 facilitates proton transfer from the sugar 4'-hydroxyl group to O(eta) of Tyr 157.

Identification and characterization of a mutation, in the human UDP-galactose-4-epimerase gene, associated with generalized epimerase-deficiency galactosemia.

Epimerase-deficiency galactosemia results from impairment of the human enzyme UDP-galactose-4-epimerase (hGALE). We and others have identified substitution mutations in the hGALE alleles of patients with the clinically mild, peripheral form of epimerase deficiency. We report here the first identification of an hGALE mutation in a patient with the clinically severe, generalized form of epimerase deficiency. The mutation, V94M, was found on both GALE alleles of this patient. This same mutation also was found in the homozygous state in two additional patients with generalized epimerase deficiency. The specific activity of the V94M-hGALE protein expressed in yeast was severely reduced with regard to UDP-galactose and partially reduced with regard to UDP-N-acetylgalactosamine. In contrast, two GALE-variant proteins associated with peripheral epimerase deficiency, L313M-hGALE and D103G-hGALE, demonstrated near-normal levels of activity with regard to both substrates, but a third allele, G90E-hGALE, demonstrated little, if any, detectable activity, despite near-normal abundance. G90E originally was identified in a heterozygous patient whose other allele remains uncharacterized. Thermal lability and protease-sensitivity studies demonstrated compromised stability in all of the partially active mutant enzymes.