UDP-glucose 4-epimerase
Encyclopedia
The enzyme
UDP-glucose 4-epimerase , also known as UDP-galactose 4-epimerase or GALE, is a homodimeric epimerase found in bacterial, plant, and mammalian cells. This enzyme performs the final step in the Leloir pathway
of galactose
metabolism, catalyzing the reversible conversion of UDP-galactose to UDP-glucose. GALE tightly binds nicotinamide adenine dinucleotide
(NAD+), a co-factor required for catalytic activity.
Additionally, human and some bacterial GALE isoforms reversibly catalyze the formation of UDP-N-acetylgalactosamine (UDP-GalNAc) from UDP-N-acetylglucosamine (UDP-GlcNAc) in the presence of NAD+, an initial step in glycoprotein
or glycolipid
synthesis.
for his discovery of sugar nucleotides and their role in the biosynthesis of carbohydrates.
scaffolds; and the ability to bind NAD+.
and humans. GALE exists as a homodimer in various species.
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. Each subunit contains two distinct domains. An N-terminal domain contains a 7-stranded parallel β-pleated sheet flanked by α-helices. Paired Rossmann fold
s within this domain allow GALE to tightly bind one NAD+ cofactor per subunit. A 6-stranded β-sheet and 5 α-helices comprise GALE's C-terminal domain. C-terminal residues bind UDP, such that the subunit is responsible for correctly positioning UDP-glucose or UDP-galactose for catalysis.
. 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, while E. coli GALE contains Tyr 149-Gly-Lys-Ser-Lys 153. The size and shape of GALE's active site varies across species, allowing for variable GALE substrate specificity. 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.
Concomitantly, the remaining 4' hydrogen is added to the si-face of NAD+, generating NADH and a 4-ketopyranose intermediate. 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. 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.
, which may be shunted into glycolysis
or the inositol
synthesis pathway.
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. Galactokinase then phosphorylates α-D-galactose at the 1' hydroxyl group, yielding galactose-1-phosphate
. 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. 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. 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. Furthermore, glucose-6-phosphate may be converted to inositol-1-phosphate by inositol-3-phosphate synthase
, generating a precursor needed for inositol
biosynthesis.
, which may exist in a mild (peripheral) or more severe (generalized) form.
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
UDP-glucose 4-epimerase , also known as UDP-galactose 4-epimerase or GALE, is a homodimeric epimerase found in bacterial, plant, and mammalian cells. This enzyme performs the final step in the Leloir pathway
Leloir pathway
The Leloir pathway is a metabolic pathway for the catabolism of D-galactose. It is named after Luis Federico Leloir.In the first step α-D-galactose is phosphorylated by a kinase to galactose 1-phosphate. Also part of this pathway is a mutarotase that facilitates the conversion of β-D-galactose to...
of galactose
Galactose
Galactose , sometimes abbreviated Gal, is a type of sugar that is less sweet than glucose. It is a C-4 epimer of glucose....
metabolism, catalyzing the reversible conversion of UDP-galactose to UDP-glucose. GALE tightly binds nicotinamide adenine dinucleotide
Nicotinamide adenine dinucleotide
Nicotinamide adenine dinucleotide, abbreviated NAD, is a coenzyme found in all living cells. The compound is a dinucleotide, since it consists of two nucleotides joined through their phosphate groups. One nucleotide contains an adenine base and the other nicotinamide.In metabolism, NAD is involved...
(NAD+), a co-factor required for catalytic activity.
Additionally, human and some bacterial GALE isoforms reversibly catalyze the formation of UDP-N-acetylgalactosamine (UDP-GalNAc) from UDP-N-acetylglucosamine (UDP-GlcNAc) in the presence of NAD+, an initial step in glycoprotein
Glycoprotein
Glycoproteins are proteins that contain oligosaccharide chains covalently attached to polypeptide side-chains. The carbohydrate is attached to the protein in a cotranslational or posttranslational modification. This process is known as glycosylation. In proteins that have segments extending...
or glycolipid
Glycolipid
Glycolipids are lipids with a carbohydrate attached. Their role is to provide energy and also serve as markers for cellular recognition.-Metabolism:...
synthesis.
Historical significance
Dr. Luis Leloir deduced the role of GALE in galactose metabolism during his tenure at the Instituto de Investigaciones Bioquímicas del Fundación Campomar, initially terming the enzyme waldenase. Dr. Leloir was awarded the 1970 Nobel Prize in ChemistryNobel Prize in Chemistry
The Nobel Prize in Chemistry is awarded annually by the Royal Swedish Academy of Sciences to scientists in the various fields of chemistry. It is one of the five Nobel Prizes established by the will of Alfred Nobel in 1895, awarded for outstanding contributions in chemistry, physics, literature,...
for his discovery of sugar nucleotides and their role in the biosynthesis of carbohydrates.
Structure
GALE belongs to the short-chain dehydrogenase/reductase (SDR) superfamily of proteins. This family is characterized by a conserved Tyr-X-X-X-Lys motif necessary for enzymatic activity; one or more Rossmann foldRossmann fold
The Rossmann fold is a protein structural motif found in proteins that bind nucleotides, especially the cofactor NAD. The structure with two repeats is composed of six parallel beta strands linked to two pairs of alpha helices in the topological order beta-alpha-beta-alpha-beta...
scaffolds; and the ability to bind NAD+.
Tertiary structure
GALE structure has been resolved for a number of species, including E. coliEscherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
and humans. GALE exists as a homodimer in various species.
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. Each subunit contains two distinct domains. An N-terminal domain contains a 7-stranded parallel β-pleated sheet flanked by α-helices. Paired Rossmann fold
Rossmann fold
The Rossmann fold is a protein structural motif found in proteins that bind nucleotides, especially the cofactor NAD. The structure with two repeats is composed of six parallel beta strands linked to two pairs of alpha helices in the topological order beta-alpha-beta-alpha-beta...
s within this domain allow GALE to tightly bind one NAD+ cofactor per subunit. A 6-stranded β-sheet and 5 α-helices comprise GALE's C-terminal domain. C-terminal residues bind UDP, such that the subunit is responsible for correctly positioning UDP-glucose or UDP-galactose for catalysis.
Active site
The cleft between GALE's N- and C-terminal domains constitutes the enzyme's active siteActive site
In biology the active site is part of an enzyme where substrates bind and undergo a chemical reaction. The majority of enzymes are proteins but RNA enzymes called ribozymes also exist. The active site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residues that...
. 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, while E. coli GALE contains Tyr 149-Gly-Lys-Ser-Lys 153. The size and shape of GALE's active site varies across species, allowing for variable GALE substrate specificity. 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.
| Residue | Function |
|---|---|
| Ala 216, Phe 218 | Anchor uracil ring to enzyme. |
| Asp 295 | Interacts with ribose 2' hydroxyl group. |
| Asn 179, Arg 231, Arg 292 | Interact with UDP phosphate groups. |
| Tyr 299, Asn 179 | Interact with galactose 2' hydroxyl or glucose 6' hydroxyl group; properly position sugar within active site. |
| Tyr 177, Phe 178 | Interact with galactose 3' hydroxyl or glucose 6' hydroxyl group; properly position sugar within active site. |
| Lys 153 | Lowers pKa of Tyr 149, allows for abstraction or donation of a hydrogen atom to or from the sugar 4' hydroxyl group. |
| Tyr 149 | Abstracts or donates a hydrogen atom to or from the sugar 4' hydroxyl group, catalyzing formation of 4-ketopyranose intermediate. |
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 hydrogen from the 4' hydroxyl group.Concomitantly, the remaining 4' hydrogen is added to the si-face of NAD+, generating NADH and a 4-ketopyranose intermediate. 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. 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.
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.Biological function
Galactose metabolism
No direct catabolic pathways exist for galactose metabolism. Galactose is therefore preferentially converted into glucose-1-phosphateGlucose-1-phosphate
Glucose 1-phosphate is a glucose molecule with a phosphate group on the 1'-carbon.-Catabolic:In glycogenolysis, it is the direct product of the reaction in which glycogen phosphorylase cleaves off a molecule of glucose from a greater glycogen structure.To be utilized in cellular catabolism it must...
, which may be shunted into glycolysis
Glycolysis
Glycolysis is the metabolic pathway that converts glucose C6H12O6, into pyruvate, CH3COCOO− + H+...
or the inositol
Inositol
Inositol or cyclohexane-1,2,3,4,5,6-hexol is a chemical compound with formula 6126 or 6, a sixfold alcohol of cyclohexane. It exists in nine possible stereoisomers, of which the most prominent form, widely occurring in nature, is cis-1,2,3,5-trans-4,6-cyclohexanehexol, or myo-inositol...
synthesis pathway.
GALE functions as one of four enzymes in the Leloir pathway
Leloir pathway
The Leloir pathway is a metabolic pathway for the catabolism of D-galactose. It is named after Luis Federico Leloir.In the first step α-D-galactose is phosphorylated by a kinase to galactose 1-phosphate. Also part of this pathway is a mutarotase that facilitates the conversion of β-D-galactose to...
of galactose conversion of glucose-1-phosphate. First, galactose mutarotase
Galactose mutarotase
Galactose mutarotase is a human enzyme that converts alpha-aldose to the beta-anomer. It belongs to family of aldose epimerases....
converts β-D-galactose to α-D-galactose. Galactokinase then phosphorylates α-D-galactose at the 1' hydroxyl group, yielding galactose-1-phosphate
Galactose-1-phosphate
Galactose-1-phosphate is an intermediate in the intraconversion of glucose and galactose.It is formed from galactose by galactokinase....
. 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. 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. 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
Glucose-6-phosphate
Glucose 6-phosphate is glucose sugar phosphorylated on carbon 6. This compound is very common in cells as the vast majority of glucose entering a cell will become phosphorylated in this way....
by phosphoglucomutase
Phosphoglucomutase
Phosphoglucomutase is an enzyme that transfers a phosphate group on an α-D-glucose monomer from the 1' to the 6' position in the forward direction or the 6' to the 1' position in the reverse direction....
. Glucose-6-phosphate readily enters glycolysis, leading to the production of ATP and pyruvate. Furthermore, glucose-6-phosphate may be converted to inositol-1-phosphate by inositol-3-phosphate synthase
Inositol-3-phosphate synthase
In enzymology, an inositol-3-phosphate synthase is an enzyme that catalyzes the chemical reactionHence, this enzyme has one substrate, D-glucose 6-phosphate, and one product, 1D-myo-inositol 3-phosphate....
, generating a precursor needed for inositol
Inositol
Inositol or cyclohexane-1,2,3,4,5,6-hexol is a chemical compound with formula 6126 or 6, a sixfold alcohol of cyclohexane. It exists in nine possible stereoisomers, of which the most prominent form, widely occurring in nature, is cis-1,2,3,5-trans-4,6-cyclohexanehexol, or myo-inositol...
biosynthesis.
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. ppGaNTase-mediated glycosylation regulates protein sorting, ligand signaling, resistance to proteolytic attack, and represents the first committed step in mucin biosynthesis.Role in disease
Human GALE deficiency or dysfunction results in Type III galactosemiaGalactosemia
Galactosemia is a rare genetic metabolic disorder that affects an individual's ability to metabolize the sugar galactose properly. Although the sugar lactose can metabolize to galactose, galactosemia is not related to and should not be confused with lactose intolerance...
, which may exist in a mild (peripheral) or more severe (generalized) form.