UDP-glucose 4-epimerase explained

UDP-glucose 4-epimerase
Ec Number:5.1.3.2
Cas Number:9032-89-7
Go Code:0003978
UDP-galactose-4-epimerase
Caption: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.
Hgncid:4116
Symbol:GALE
Entrezgene:2582
Omim:606953
Refseq:NM_000403
Uniprot:Q14376
Ecnumber:5.1.3.2
Chromosome:1
Arm:p
Band:36
Locussupplementarydata:-p35

The enzyme UDP-glucose 4-epimerase, 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]

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]

Residue! width="600"
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.

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] [9]

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.[9] 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.[10]

Biological function

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.[11]

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.[12] Furthermore, glucose-6-phosphate may be converted to inositol-1-phosphate by inositol-3-phosphate synthase, generating a precursor needed for inositol biosynthesis.[13]

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.[14] ppGaNTase-mediated glycosylation regulates protein sorting,[15] [16] [17] [18] [19] ligand signaling,[20] [21] [22] resistance to proteolytic attack,[23] [24] and represents the first committed step in mucin biosynthesis.[14]

Role in disease

See main article: 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.[11]

Further reading

External links

Notes and References

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

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