UDP-glucose 4-epimerase

Last updated
UDP-glucose 4-epimerase
Identifiers
Aliases UDPgalactose 4-epimerase4-epimeraseuridine diphosphate glucose 4-epimeraseUDPG-4-epimeraseUDP-galactose 4-epimeraseuridine diphosphoglucose epimeraseuridine diphospho-galactose-4-epimeraseUDP-D-galactose 4-epimeraseUDP-glucose epimeraseuridine diphosphoglucose 4-epimeraseuridine diphosphate galactose 4-epimerase
External IDs GeneCards:
Orthologs
SpeciesHumanMouse
Entrez
Ensembl
UniProt
RefSeq (mRNA)

n/a

n/a

RefSeq (protein)

n/a

n/a

Location (UCSC)n/an/a
PubMed searchn/an/a
Wikidata
View/Edit Human
UDP-glucose 4-epimerase
Human GALE bound to NADH and UDP-glucose.png
H. sapiens UDP-glucose 4-epimerase homodimer bound to NADH and UDP-glucose. Domains: N-terminal and C-terminal.
Identifiers
EC no. 5.1.3.2
CAS no. 9032-89-7
Databases
IntEnz IntEnz view
BRENDA BRENDA entry
ExPASy NiceZyme view
KEGG KEGG entry
MetaCyc metabolic pathway
PRIAM profile
PDB structures RCSB PDB PDBe PDBsum
Gene Ontology AmiGO / QuickGO
UDP-galactose-4-epimerase
Human GALE bound to NAD+ and UDP-GlcNAc.png
Human GALE bound to NAD+ and UDP-GlcNAc, with N- and C-terminal domains highlighted. Asn 207 contorts to accommodate UDP-GlcNAc within the active site.
Identifiers
SymbolGALE
NCBI gene 2582
HGNC 4116
OMIM 606953
RefSeq NM_000403
UniProt Q14376
Other data
EC number 5.1.3.2
Locus Chr. 1 p36-p35
NAD-dependent epimerase/dehydratase
Identifiers
Symbol?
Pfam PF01370
InterPro IPR001509
Membranome 330

The enzyme UDP-glucose 4-epimerase (EC 5.1.3.2), also known as UDP-galactose 4-epimerase or GALE, is a homodimeric epimerase found in bacterial, fungal, 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. [1] GALE tightly binds nicotinamide adenine dinucleotide (NAD+), a co-factor required for catalytic activity. [2]

Contents

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. [3]

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. [4] Dr. Leloir was awarded the 1970 Nobel Prize in Chemistry for his discovery of sugar nucleotides and their role in the biosynthesis of carbohydrates. [5]

Structure

GALE belongs to the short-chain dehydrogenase/reductase (SDR) superfamily of proteins. [6] This family is characterized by a conserved Tyr-X-X-X-Lys motif necessary for enzymatic activity; one or more Rossmann fold scaffolds; and the ability to bind NAD+. [6]

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]

Known E. coli GALE residue interactions with UDP-glucose and UDP-galactose. [9]
ResidueFunction
Ala 216, Phe 218Anchor uracil ring to enzyme.
Asp 295Interacts with ribose 2' hydroxyl group.
Asn 179, Arg 231, Arg 292Interact with UDP phosphate groups.
Tyr 299, Asn 179Interact with galactose 2' hydroxyl or glucose 6' hydroxyl group; properly position sugar within active site.
Tyr 177, Phe 178Interact with galactose 3' hydroxyl or glucose 6' hydroxyl group; properly position sugar within active site.
Lys 153Lowers pKa of Tyr 149, allows for abstraction or donation of a hydrogen atom to or from the sugar 4' hydroxyl group.
Tyr 149Abstracts or donates a hydrogen atom to or from the sugar 4' hydroxyl group, catalyzing formation of 4-ketopyranose intermediate.

Mechanism

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]

Biological function

Intermediates and enzymes in the Leloir pathway of galactose metabolism. Leloir pathway.png
Intermediates and enzymes in the Leloir pathway of galactose metabolism.

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]

Role in disease

Human GALE deficiency or dysfunction results in Type III galactosemia, which may exist in a mild (peripheral) or more severe (generalized) form. [12]

Related Research Articles

Galactose Monosaccharide sugar

Galactose sometimes abbreviated Gal, is a monosaccharide sugar that is about as sweet as glucose, and about 65% as sweet as sucrose. It is an aldohexose and a C-4 epimer of glucose. A galactose molecule linked with a glucose molecule forms a lactose molecule.

Galactokinase

Galactokinase is an enzyme (phosphotransferase) that facilitates the phosphorylation of α-D-galactose to galactose 1-phosphate at the expense of one molecule of ATP. Galactokinase catalyzes the second step of the Leloir pathway, a metabolic pathway found in most organisms for the catabolism of α-D-galactose to glucose 1-phosphate. First isolated from mammalian liver, galactokinase has been studied extensively in yeast, archaea, plants, and humans.

Galactose-1-phosphate uridylyltransferase Mammalian protein found in Homo sapiens

Galactose-1-phosphate uridylyltransferase is an enzyme responsible for converting ingested galactose to glucose.

Glycosyltransferase Class of enzymes that catalyze the transfer of glycosyl groups to an acceptor

Glycosyltransferases are enzymes that establish natural glycosidic linkages. They catalyze the transfer of saccharide moieties from an activated nucleotide sugar to a nucleophilic glycosyl acceptor molecule, the nucleophile of which can be oxygen- carbon-, nitrogen-, or sulfur-based.

UTP—glucose-1-phosphate uridylyltransferase

UTP—glucose-1-phosphate uridylyltransferase also known as glucose-1-phosphate uridylyltransferase is an enzyme involved in carbohydrate metabolism. It synthesizes UDP-glucose from glucose-1-phosphate and UTP; i.e.,

Galactose epimerase deficiency Medical condition

Galactose epimerase deficiency, also known as GALE deficiency, Galactosemia III and UDP-galactose-4-epimerase deficiency, is a rare, autosomal recessive form of galactosemia associated with a deficiency of the enzyme galactose epimerase.

Galactose-1-phosphate uridylyltransferase deficiency Medical condition

Galactose-1-phosphate uridylyltransferase deficiency(classic galactosemia) is the most common type of galactosemia, an inborn error of galactose metabolism, caused by a deficiency of the enzyme galactose-1-phosphate uridylyltransferase. It is an autosomal recessive metabolic disorder that can cause liver disease and death if untreated. Treatment of galactosemia is most successful if initiated early and includes dietary restriction of lactose intake. Because early intervention is key, galactosemia is included in newborn screening programs in many areas. On initial screening, which often involves measuring the concentration of galactose in blood, classic galactosemia may be indistinguishable from other inborn errors of galactose metabolism, including galactokinase deficiency and galactose epimerase deficiency. Further analysis of metabolites and enzyme activities are needed to identify the specific metabolic error.

Tunicamycin Chemical compound

Tunicamycin is a mixture of homologous nucleoside antibiotics that inhibits the UDP-HexNAc: polyprenol-P HexNAc-1-P family of enzymes. In eukaryotes, this includes the enzyme GlcNAc phosphotransferase (GPT), which catalyzes the transfer of N-acetylglucosamine-1-phosphate from UDP-N-acetylglucosamine to dolichol phosphate in the first step of glycoprotein synthesis. Tunicamycin blocks N-linked glycosylation (N-glycans) and treatment of cultured human cells with tunicamycin causes cell cycle arrest in G1 phase. It is used as an experimental tool in biology, e.g. to induce unfolded protein response. Tunicamycin is produced by several bacteria, including Streptomyces clavuligerus and Streptomyces lysosuperificus.

UDP-N-acetylglucosamine 2-epimerase

In enzymology, an UDP-N-acetylglucosamine 2-epimerase is an enzyme that catalyzes the chemical reaction

Nucleotide sugars are the activated forms of monosaccharides. Nucleotide sugars act as glycosyl donors in glycosylation reactions. Those reactions are catalyzed by a group of enzymes called glycosyltransferases.

In enzymology, a N-acetylgalactosamine kinase is an enzyme that catalyzes the chemical reaction

UDP-glucose—hexose-1-phosphate uridylyltransferase

In enzymology, an UDP-glucose—hexose-1-phosphate uridylyltransferase is an enzyme that catalyzes the chemical reaction

B4GALT1 Protein-coding gene in the species Homo sapiens

Beta-1,4-galactosyltransferase 1 is an enzyme that in humans is encoded by the B4GALT1 gene.

GALNT1

Polypeptide N-acetylgalactosaminyltransferase 1 is an enzyme that in humans is encoded by the GALNT1 gene.

GALNT2 Protein-coding gene in the species Homo sapiens

Polypeptide N-acetylgalactosaminyltransferase 2 is an enzyme that in humans is encoded by the GALNT2 gene.

B3GALT5 Protein-coding gene in the species Homo sapiens

Beta-1,3-galactosyltransferase 5 is an enzyme that in humans is encoded by the B3GALT5 gene.

The gal operon is a prokaryotic operon, which encodes enzymes necessary for galactose metabolism. Repression of gene expression for this operon works via binding of repressor molecules to two operators. These repressors dimerize, creating a loop in the DNA. The loop as well as hindrance from the external operator prevent RNA polymerase from binding to the promoter, and thus prevent transcription. Additionally, since the metabolism of galactose in the cell is involved in both anabolic and catabolic pathways, a novel regulatory system using two promoters for differential repression has been identified and characterized within the context of the gal operon.

Inborn errors of carbohydrate metabolism are inborn error of metabolism that affect the catabolism and anabolism of carbohydrates.

Galactolysis refers to the catabolism of galactose.

Leloir pathway

The Leloir pathway is a metabolic pathway for the catabolism of D-galactose. It is named after Luis Federico Leloir, who first described it.

References

  1. 1 2 3 4 5 6 7 8 9 10 11 Holden HM, Rayment I, Thoden JB (November 2003). "Structure and function of enzymes of the Leloir pathway for galactose metabolism". J. Biol. Chem. 278 (45): 43885–8. doi: 10.1074/jbc.R300025200 . PMID   12923184.
  2. 1 2 Liu Y, Vanhooke JL, Frey PA (June 1996). "UDP-galactose 4-epimerase: NAD+ content and a charge-transfer band associated with the substrate-induced conformational transition". Biochemistry. 35 (23): 7615–20. doi:10.1021/bi960102v. PMID   8652544.
  3. 1 2 3 4 Thoden JB, Wohlers TM, Fridovich-Keil JL, Holden HM (May 2001). "Human UDP-galactose 4-epimerase. Accommodation of UDP-N-acetylglucosamine within the active site". J. Biol. Chem. 276 (18): 15131–6. doi: 10.1074/jbc.M100220200 . PMID   11279032.
  4. LELOIR LF (September 1951). "The enzymatic transformation of uridine diphosphate glucose into a galactose derivative". Arch Biochem. 33 (2): 186–90. doi:10.1016/0003-9861(51)90096-3. hdl: 11336/140700 . PMID   14885999.
  5. "The Nobel Prize in Chemistry 1970" (Press release). The Royal Swedish Academy of Science. 1970. Retrieved 2010-05-17.
  6. 1 2 3 4 Kavanagh KL, Jörnvall H, Persson B, Oppermann U (December 2008). "Medium- and short-chain dehydrogenase/reductase gene and protein families : the SDR superfamily: functional and structural diversity within a family of metabolic and regulatory enzymes". Cell. Mol. Life Sci. 65 (24): 3895–906. doi:10.1007/s00018-008-8588-y. PMC   2792337 . PMID   19011750.
  7. 1 2 PDB: 1EK5 ; Thoden JB, Wohlers TM, Fridovich-Keil JL, Holden HM (May 2000). "Crystallographic evidence for Tyr 157 functioning as the active site base in human UDP-galactose 4-epimerase". Biochemistry. 39 (19): 5691–701. doi:10.1021/bi000215l. PMID   10801319.
  8. 1 2 3 PDB: 1XEL ; Thoden JB, Frey PA, Holden HM (April 1996). "Molecular structure of the NADH/UDP-glucose abortive complex of UDP-galactose 4-epimerase from Escherichia coli: implications for the catalytic mechanism". Biochemistry. 35 (16): 5137–44. doi:10.1021/bi9601114. PMID   8611497.
  9. PDB: 1A9Z ; Thoden JB, Holden HM (August 1998). "Dramatic differences in the binding of UDP-galactose and UDP-glucose to UDP-galactose 4-epimerase from Escherichia coli". Biochemistry. 37 (33): 11469–77. doi:10.1021/bi9808969. PMID   9708982.
  10. 1 2 Liu Y, Thoden JB, Kim J, Berger E, Gulick AM, Ruzicka FJ, Holden HM, Frey PA (September 1997). "Mechanistic roles of tyrosine 149 and serine 124 in UDP-galactose 4-epimerase from Escherichia coli". Biochemistry. 36 (35): 10675–84. doi:10.1021/bi970430a. PMID   9271498.
  11. Kingsley DM, Kozarsky KF, Hobbie L, Krieger M (March 1986). "Reversible defects in O-linked glycosylation and LDL receptor expression in a UDP-Gal/UDP-GalNAc 4-epimerase deficient mutant". Cell. 44 (5): 749–59. doi:10.1016/0092-8674(86)90841-X. PMID   3948246. S2CID   28293937.
  12. 1 2 Lai K, Elsas LJ, Wierenga KJ (November 2009). "Galactose toxicity in animals". IUBMB Life. 61 (11): 1063–74. doi:10.1002/iub.262. PMC   2788023 . PMID   19859980.
  13. Stryer, Lubert; Berg, Jeremy Mark; Tymoczko, John L. (2008). Biochemistry (Looseleaf) . San Francisco: W. H. Freeman. pp.  443–58. ISBN   9780716718437.
  14. Michell RH (February 2008). "Inositol derivatives: evolution and functions". Nat. Rev. Mol. Cell Biol. 9 (2): 151–61. doi:10.1038/nrm2334. PMID   18216771. S2CID   3245927.
  15. 1 2 Ten Hagen KG, Fritz TA, Tabak LA (January 2003). "All in the family: the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases". Glycobiology. 13 (1): 1R–16R. doi: 10.1093/glycob/cwg007 . PMID   12634319.
  16. Alfalah M, Jacob R, Preuss U, Zimmer KP, Naim H, Naim HY (June 1999). "O-linked glycans mediate apical sorting of human intestinal sucrase-isomaltase through association with lipid rafts". Curr. Biol. 9 (11): 593–6. doi: 10.1016/S0960-9822(99)80263-2 . PMID   10359703. S2CID   16866875.
  17. Altschuler Y, Kinlough CL, Poland PA, Bruns JB, Apodaca G, Weisz OA, Hughey RP (March 2000). "Clathrin-mediated endocytosis of MUC1 is modulated by its glycosylation state". Mol. Biol. Cell. 11 (3): 819–31. doi:10.1091/mbc.11.3.819. PMC   14813 . PMID   10712502.
  18. Breuza L, Garcia M, Delgrossi MH, Le Bivic A (February 2002). "Role of the membrane-proximal O-glycosylation site in sorting of the human receptor for neurotrophins to the apical membrane of MDCK cells". Exp. Cell Res. 273 (2): 178–86. doi:10.1006/excr.2001.5442. PMID   11822873.
  19. Naim HY, Joberty G, Alfalah M, Jacob R (June 1999). "Temporal association of the N- and O-linked glycosylation events and their implication in the polarized sorting of intestinal brush border sucrase-isomaltase, aminopeptidase N, and dipeptidyl peptidase IV". J. Biol. Chem. 274 (25): 17961–7. doi: 10.1074/jbc.274.25.17961 . PMID   10364244.
  20. Zheng X, Sadler JE (March 2002). "Mucin-like domain of enteropeptidase directs apical targeting in Madin-Darby canine kidney cells". J. Biol. Chem. 277 (9): 6858–63. doi: 10.1074/jbc.M109857200 . PMID   11878264.
  21. Hooper LV, Gordon JI (February 2001). "Glycans as legislators of host-microbial interactions: spanning the spectrum from symbiosis to pathogenicity". Glycobiology. 11 (2): 1R–10R. doi: 10.1093/glycob/11.2.1R . PMID   11287395.
  22. Yeh JC, Hiraoka N, Petryniak B, Nakayama J, Ellies LG, Rabuka D, Hindsgaul O, Marth JD, Lowe JB, Fukuda M (June 2001). "Novel sulfated lymphocyte homing receptors and their control by a Core1 extension beta 1,3-N-acetylglucosaminyltransferase". Cell. 105 (7): 957–69. doi: 10.1016/S0092-8674(01)00394-4 . PMID   11439191. S2CID   18674112.
  23. Somers WS, Tang J, Shaw GD, Camphausen RT (October 2000). "Insights into the molecular basis of leukocyte tethering and rolling revealed by structures of P- and E-selectin bound to SLe(X) and PSGL-1". Cell. 103 (3): 467–79. doi: 10.1016/S0092-8674(00)00138-0 . PMID   11081633. S2CID   12719907.
  24. Sauer J, Sigurskjold BW, Christensen U, Frandsen TP, Mirgorodskaya E, Harrison M, Roepstorff P, Svensson B (December 2000). "Glucoamylase: structure/function relationships, and protein engineering". Biochim. Biophys. Acta. 1543 (2): 275–293. doi:10.1016/s0167-4838(00)00232-6. PMID   11150611.
  25. Garner B, Merry AH, Royle L, Harvey DJ, Rudd PM, Thillet J (June 2001). "Structural elucidation of the N- and O-glycans of human apolipoprotein(a): role of o-glycans in conferring protease resistance". J. Biol. Chem. 276 (25): 22200–8. doi: 10.1074/jbc.M102150200 . PMID   11294842.

Further reading