UDP-glucose 4-epimerase

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.{{cite journal | author = LELOIR LF | title = The enzymatic transformation of uridine diphosphate glucose into a galactose derivative | journal = Arch Biochem | volume = 33 | issue = 2 | pages = 186–90 |date=September 1951 | pmid = 14885999 | doi = 10.1016/0003-9861(51)90096-3| hdl = 11336/140700 | hdl-access = free }} Dr. Leloir was awarded the 1970 Nobel Prize in Chemistry for his discovery of sugar nucleotides and their role in the biosynthesis of carbohydrates.{{cite press release | url = http://nobelprize.org/nobel_prizes/chemistry/laureates/1970/press.html | title=The Nobel Prize in Chemistry 1970 | publisher = The Royal Swedish Academy of Science | year = 1970 | access-date = 2010-05-17}}

GALE belongs to the short-chain dehydrogenase/reductase (SDR) superfamily of proteins.{{cite journal |vauthors=Kavanagh KL, Jörnvall H, Persson B, Oppermann U | title = 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 | journal = Cell. Mol. Life Sci. | volume = 65 | issue = 24 | pages = 3895–906 |date=December 2008 | pmid = 19011750 | pmc = 2792337 | doi = 10.1007/s00018-008-8588-y }} 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⁺.

GALE structure has been resolved for a number of species, including E. coli{{PDB|1EK5}}; {{cite journal |vauthors=Thoden JB, Wohlers TM, Fridovich-Keil JL, Holden HM | title = Crystallographic evidence for Tyr 157 functioning as the active site base in human UDP-galactose 4-epimerase | journal = Biochemistry | volume = 39 | issue = 19 | pages = 5691–701 |date=May 2000 | pmid = 10801319 | doi = 10.1021/bi000215l}} and humans.{{PDB|1XEL}}; {{cite journal |vauthors=Thoden JB, Frey PA, Holden HM | title = Molecular structure of the NADH/UDP-glucose abortive complex of UDP-galactose 4-epimerase from Escherichia coli: implications for the catalytic mechanism | journal = Biochemistry | volume = 35 | issue = 16 | pages = 5137–44 |date=April 1996 | pmid = 8611497 | doi = 10.1021/bi9601114 }} GALE exists as a homodimer in various species.

While subunit size varies from 68 amino acids [https://www.ncbi.nlm.nih.gov/protein/ZP_05580029.1 (Enterococcus faecalis)] to 564 amino acids [https://www.ncbi.nlm.nih.gov/protein/YP_701629.1 (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 folds 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.

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, 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.

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.{{cite journal |vauthors=Liu Y, Thoden JB, Kim J, Berger E, Gulick AM, Ruzicka FJ, Holden HM, Frey PA | title = Mechanistic roles of tyrosine 149 and serine 124 in UDP-galactose 4-epimerase from Escherichia coli | journal = Biochemistry | volume = 36 | issue = 35 | pages = 10675–84 |date=September 1997 | pmid = 9271498 | doi = 10.1021/bi970430a }}

Concomitantly, the 4' hydride 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.

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.{{cite journal |vauthors=Kingsley DM, Kozarsky KF, Hobbie L, Krieger M | title = Reversible defects in O-linked glycosylation and LDL receptor expression in a UDP-Gal/UDP-GalNAc 4-epimerase deficient mutant | journal = Cell | volume = 44 | issue = 5 | pages = 749–59 |date=March 1986 | pmid = 3948246 | doi = 10.1016/0092-8674(86)90841-X| s2cid = 28293937 }}

File:Leloir pathway.png

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.{{cite journal |vauthors=Lai K, Elsas LJ, Wierenga KJ | title = Galactose toxicity in animals | journal = IUBMB Life | volume = 61 | issue = 11 | pages = 1063–74 |date=November 2009 | pmid = 19859980 | pmc = 2788023 | doi = 10.1002/iub.262 }}

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.{{cite book | author1 = Stryer, Lubert | author2 = Berg, Jeremy Mark | author3 = Tymoczko, John L. | title = Biochemistry (Looseleaf) | publisher = W. H. Freeman | location = San Francisco | year = 2008 | pages = [https://archive.org/details/biochemistry3rdedi00stry/page/443 443–58] | isbn = 9780716718437 | url = https://archive.org/details/biochemistry3rdedi00stry/page/443 | url-access = registration }} Furthermore, glucose-6-phosphate may be converted to inositol-1-phosphate by inositol-3-phosphate synthase, generating a precursor needed for inositol biosynthesis.{{cite journal | author = Michell RH | title = Inositol derivatives: evolution and functions | journal = Nat. Rev. Mol. Cell Biol. | volume = 9 | issue = 2 | pages = 151–61 |date=February 2008 | pmid = 18216771 | doi = 10.1038/nrm2334 | s2cid = 3245927 }}

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.{{cite journal |vauthors=Ten Hagen KG, Fritz TA, Tabak LA | title = All in the family: the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases | journal = Glycobiology | volume = 13 | issue = 1 | pages = 1R–16R |date=January 2003 | pmid = 12634319 | doi = 10.1093/glycob/cwg007 | doi-access = free }} ppGaNTase-mediated glycosylation regulates protein sorting,{{cite journal |vauthors=Alfalah M, Jacob R, Preuss U, Zimmer KP, Naim H, Naim HY | title = O-linked glycans mediate apical sorting of human intestinal sucrase-isomaltase through association with lipid rafts | journal = Curr. Biol. | volume = 9 | issue = 11 | pages = 593–6 |date=June 1999 | pmid = 10359703 | doi = 10.1016/S0960-9822(99)80263-2| s2cid = 16866875 | doi-access = free | bibcode = 1999CBio....9..593A }}{{cite journal |vauthors=Altschuler Y, Kinlough CL, Poland PA, Bruns JB, Apodaca G, Weisz OA, Hughey RP | title = Clathrin-mediated endocytosis of MUC1 is modulated by its glycosylation state | journal = Mol. Biol. Cell | volume = 11 | issue = 3 | pages = 819–31 |date=March 2000 | pmid = 10712502 | pmc = 14813 | doi = 10.1091/mbc.11.3.819}}{{cite journal |vauthors=Breuza L, Garcia M, Delgrossi MH, Le Bivic A | title = Role of the membrane-proximal O-glycosylation site in sorting of the human receptor for neurotrophins to the apical membrane of MDCK cells | journal = Exp. Cell Res. | volume = 273 | issue = 2 | pages = 178–86 |date=February 2002 | pmid = 11822873 | doi = 10.1006/excr.2001.5442 }}{{cite journal |vauthors=Naim HY, Joberty G, Alfalah M, Jacob R | title = 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 | journal = J. Biol. Chem. | volume = 274 | issue = 25 | pages = 17961–7 |date=June 1999 | pmid = 10364244 | doi = 10.1074/jbc.274.25.17961| doi-access = free }}{{cite journal|author-link2=J. Evan Sadler |vauthors=Zheng X, Sadler JE | title = Mucin-like domain of enteropeptidase directs apical targeting in Madin-Darby canine kidney cells | journal = J. Biol. Chem. | volume = 277 | issue = 9 | pages = 6858–63 |date=March 2002 | pmid = 11878264 | doi = 10.1074/jbc.M109857200| doi-access = free }} ligand signaling,{{cite journal |vauthors=Hooper LV, Gordon JI | title = Glycans as legislators of host-microbial interactions: spanning the spectrum from symbiosis to pathogenicity | journal = Glycobiology | volume = 11 | issue = 2 | pages = 1R–10R |date=February 2001 | pmid = 11287395 | doi = 10.1093/glycob/11.2.1R| doi-access = free }}{{cite journal |vauthors=Yeh JC, Hiraoka N, Petryniak B, Nakayama J, Ellies LG, Rabuka D, Hindsgaul O, Marth JD, Lowe JB, Fukuda M | title = Novel sulfated lymphocyte homing receptors and their control by a Core1 extension beta 1,3-N-acetylglucosaminyltransferase | journal = Cell | volume = 105 | issue = 7 | pages = 957–69 |date=June 2001 | pmid = 11439191 | doi = 10.1016/S0092-8674(01)00394-4| s2cid = 18674112 | doi-access = free }}{{cite journal |vauthors=Somers WS, Tang J, Shaw GD, Camphausen RT | title = 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 | journal = Cell | volume = 103 | issue = 3 | pages = 467–79 |date=October 2000 | pmid = 11081633 | doi = 10.1016/S0092-8674(00)00138-0| s2cid = 12719907 | doi-access = free }} resistance to proteolytic attack,{{cite journal |vauthors=Sauer J, Sigurskjold BW, Christensen U, Frandsen TP, Mirgorodskaya E, Harrison M, Roepstorff P, Svensson B | title = Glucoamylase: structure/function relationships, and protein engineering | journal = Biochim. Biophys. Acta | volume = 1543 | issue = 2 | pages = 275–293 |date=December 2000 | pmid = 11150611 | doi = 10.1016/s0167-4838(00)00232-6}}{{cite journal |vauthors=Garner B, Merry AH, Royle L, Harvey DJ, Rudd PM, Thillet J | title = Structural elucidation of the N- and O-glycans of human apolipoprotein(a): role of o-glycans in conferring protease resistance | journal = J. Biol. Chem. | volume = 276 | issue = 25 | pages = 22200–8 |date=June 2001 | pmid = 11294842 | doi = 10.1074/jbc.M102150200 | doi-access = free }} and represents the first committed step in mucin biosynthesis.

{{main|Galactose epimerase deficiency}}

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

{{reflist|2}}

{{refbegin}}

• {{cite book | author = Leloir LF | chapter = Enzymic Isomerization and Related Processes | title = Advances in Enzymology and Related Areas of Molecular Biology | year = 1953 | journal = Adv. Enzymol. Relat. Subj. Biochem. | volume = 14 | pages = 193–218 | pmid = 13057717 | doi = 10.1002/9780470122594.ch6 | series = Advances in Enzymology - and Related Areas of Molecular Biology | isbn = 9780470122594 }}
• {{cite journal |vauthors=Maxwell ES, de Robichon-Szulmajster H| year = 1960 | title = Purification of uridine diphosphate galactose-4-epimerase from yeast and the identification of protein-bound diphosphopyridine nucleotide | journal = J. Biol. Chem. | volume = 235 | issue = 2 | pages = 308–312 | doi = 10.1016/S0021-9258(18)69520-1 | doi-access = free }}
• {{cite journal |vauthors=Wilson DB, Hogness DS| title = The enzymes of the galactose operon in Escherichia coli. I Purification and characterization of uridine diphosphogalactose 4-epimerase | journal = J. Biol. Chem. | volume = 239 | pages = 2469–81 |date=August 1964 | doi = 10.1016/S0021-9258(18)93876-7 | pmid = 14235524 | doi-access = free }}

{{refend}}

• [https://www.ncbi.nlm.nih.gov/books/NBK51671/ GeneReviews/NCBI/NIH/UW entry on Epimerase Deficiency Galactosemia]
• [https://www.ncbi.nlm.nih.gov/omim/230350,606953,230350,606953 OMIM entries on Epimerase Deficiency Galactosemia]
• {{MeSH name|UDPgalactose+4-Epimerase}}

{{Fructose and galactose metabolism enzymes}}

{{Racemases and epimerases}}

{{Enzymes}}

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Category:EC 5.1.3

Category:Enzymes of known structure

Category:NADH-dependent enzymes