Tertiary structure

GALE structure has been resolved for a number of species, including E. coli^[7] and humans.^[8] GALE exists as a homodimer in various species.^[8]

While subunit size varies from 68 amino acids (Enterococcus faecalis) to 564 amino acids (Rhodococcus jostii), a majority of GALE subunits cluster near 330 amino acids in length.^[6] Each subunit contains two distinct domains. An N-terminal domain contains a 7-stranded parallel β-pleated sheet flanked by α-helices.^[1] Paired Rossmann folds within this domain allow GALE to tightly bind one NAD⁺ cofactor per subunit.^[2] A 6-stranded β-sheet and 5 α-helices comprise GALE's C-terminal domain.^[1] C-terminal residues bind UDP, such that the subunit is responsible for correctly positioning UDP-glucose or UDP-galactose for catalysis.^[1]

Active site

The cleft between GALE's N- and C-terminal domains constitutes the enzyme's active site. A conserved Tyr-X-X-X Lys motif is necessary for GALE catalytic activity; in humans, this motif is represented by Tyr 157-Gly-Lys-Ser-Lys 161,^[6] while E. coli GALE contains Tyr 149-Gly-Lys-Ser-Lys 153.^[8] The size and shape of GALE's active site varies across species, allowing for variable GALE substrate specificity.^[3] Additionally, the conformation of the active site within a species-specific GALE is malleable; for instance, a bulky UDP-GlcNAc 2' N-acetyl group is accommodated within the human GALE active site by the rotation of the Asn 207 carboxamide side chain.^[3]

Conversion of UDP-galactose to UDP-glucose

GALE inverts the configuration of the 4' hydroxyl group of UDP-galactose through a series of 4 steps. Upon binding UDP-galactose, a conserved tyrosine residue in the active site abstracts a proton from the 4' hydroxyl group.^[7][10]

Concomitantly, the 4' hydride is added to the si-face of NAD+, generating NADH and a 4-ketopyranose intermediate.^[1] The 4-ketopyranose intermediate rotates 180° about the pyrophosphoryl linkage between the glycosyl oxygen and β-phosphorus atom, presenting the opposite face of the ketopyranose intermediate to NADH.^[10] Hydride transfer from NADH to this opposite face inverts the stereochemistry of the 4' center. The conserved tyrosine residue then donates its proton, regenerating the 4' hydroxyl group.^[1]

Conversion of UDP-GlcNAc to UDP-GalNAc

Human and some bacterial GALE isoforms reversibly catalyze the conversion of UDP-GlcNAc to UDP-GalNAc through an identical mechanism, inverting the stereochemical configuration at the sugar's 4' hydroxyl group.^[3][11]

Galactose metabolism

No direct catabolic pathways exist for galactose metabolism. Galactose is therefore preferentially converted into glucose-1-phosphate, which may be shunted into glycolysis or the inositol synthesis pathway.^[12]

GALE functions as one of four enzymes in the Leloir pathway of galactose conversion of glucose-1-phosphate. First, galactose mutarotase converts β-D-galactose to α-D-galactose.^[1] Galactokinase then phosphorylates α-D-galactose at the 1' hydroxyl group, yielding galactose-1-phosphate.^[1] In the third step, galactose-1-phosphate uridyltransferase catalyzes the reversible transfer of a UMP moiety from UDP-glucose to galactose-1-phosphate, generating UDP-galactose and glucose-1-phosphate.^[1] In the final Leloir step, UDP-glucose is regenerated from UDP-galactose by GALE; UDP-glucose cycles back to the third step of the pathway.^[1] As such, GALE regenerates a substrate necessary for continued Leloir pathway cycling.

The glucose-1-phosphate generated in step 3 of the Leloir pathway may be isomerized to glucose-6-phosphate by phosphoglucomutase. Glucose-6-phosphate readily enters glycolysis, leading to the production of ATP and pyruvate.^[13] Furthermore, glucose-6-phosphate may be converted to inositol-1-phosphate by inositol-3-phosphate synthase, generating a precursor needed for inositol biosynthesis.^[14]

UDP-GalNAc synthesis

Human and selected bacterial GALE isoforms bind UDP-GlcNAc, reversibly catalyzing its conversion to UDP-GalNAc. A family of glycosyltransferases known as UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosamine transferases (ppGaNTases) transfers GalNAc from UDP-GalNAc to glycoprotein serine and threonine residues.^[15] ppGaNTase-mediated glycosylation regulates protein sorting,^{[16][17][18][19][20]} ligand signaling,^[21][22][23] resistance to proteolytic attack,^[24][25] and represents the first committed step in mucin biosynthesis.^[15]