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Glycobiology 8130,Lecture 220/1/2009

glycan precursors:NDP-sugar synthesis and

transporters

Maor Bar-PeledCCRC

Peled@ccrc.uga.edu

Most glycan are faced outside a cell

• Many structures of glycans. Methods in Enzymology Vol 405; 2005

Glycoprotein N-glycosylation Glycoprotein O-glycosylation

O-FucO-ManO-Glc

GlycosylphosphatidylinositolProtein (GPI)

Sphingosines

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Synthesis of a glycan requires co-ordinateactivities of multiple cellular systems

• Yusuke Maeda, Hisashi Ashida and Taroh Kinoshita 2006 synthesis ofGPI anchor . Meth Enz 416:182-205

History

• ~100 years ago (1906) Harden and Yodiscovered that yeast fermented with sugar accumulate a

phosphate esters-sugar derivative. Man-6-P, Glc-6-P･ the synthesis, and metabolic roles of sugar-phosphates

start to emerge.･The synthesis of a polysaccharide from glucose-1-phosphate in

muscle extract. Carl F. Cori, Gerhard Schmidt, and Gerty T. CoriScience 19 May 1939

1947 Nobel Prize. Coriʼs(Glycogen synthesis, metabolism& disease)

EMIL FISCHER observed in 1890 that L-glucose was not metabolizedby brewer' yeast

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Building block for glycansynthesis

• Nucleotide-sugars• Other activated-sugars

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Glycans are made from activated sugars

• 1950- the discovery of nucleotide-sugars.Sugar-donor for glycan synthesis

Hehre 1941 SCIENCE------SPECIAL ARTICLES------Sucrose incorporated into bacterial polysaccharide.

1946,1947-- Glc-1-P is involved….. C. diphtheria starch-like

With collaborators, Ranwel Caputto, Carlos E.Cardini, Raúl Trucco and Alejandro C. Paladinistudythe metabolism of galactose which led tothe isolation of glucose 1,6-diphosphate anduridine diphosphate glucose.

UDP-gluocse served asglucose donor in thesynthesis of trehalose(with Enrico Cabib, 1953 )and sucrose (with CarlosE. Cardini andJ.Chiriboga, 1955) 1970, Nobel Proze

Nucleotide-sugar

• There are many types of nucleotide sugars.– Fungi 10-15– Human 10– Plant >25– Bacteria >70

• The nucleotide can be ADP, GDP, CDP, TDP, UDP,CMP

Sugar Nucleotide

PO

O

P

O

O

O-

1

23

4

O

OHHO

5

HO

CH2OH

O CH25ʼ O

OHOH

HN

O

O N1

2

3 45

6

2ʼ3ʼ

4ʼ

u

rib

O-

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A Glycosyl-transferase uses a nucleotide-sugars to build a glycan

Glycans are made from activated sugars• Glycosyltransferases (GT) are a group of enzymes that

transfer of a sugar moiety from an activated sugar ontosaccharide or non-saccharide acceptors.

• GT attaches the sugar in a specific linkage (eg alpha, beta;1-2 , 1-4)

--Over 7,200 of GT-related sequences inCAZY database.Most GT, but not allare using NDP-sugar as donors

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Role of a nucleotide sugars in biology• Nucleotide-sugars are precursors for synthesis of macromolecules:

– Glycoproteins– Glycolipids,

• Lipopolysaccharides..– Proteoglycans

• heparan– Oligosaccharides

• Raffinose..– Polysaccharides

• Starch, cellulose, pectin, xylan (biomass……biofuel)

• Nucleotide-sugars are precursors for synthesis of small molecules:– Toxins,

• Liver toxin removal– Antibiotics,

• Kanamycin, neomycin– Smaller metabolites,

• Hormones, bitter, colors, storage:sucrose, trehalose, lactose, maltose– Hormones,

Synthesis of other activated -sugars1- dolichol-P[P]-sugars (e.g. Dol-P-Man)

2. Isoprenol-PP-sugar (e.g. Dodecaprenyl phosphate-galacturonic acid)

3. Park nucleotides

4. CMP-sugars (e.g. CMP-Sialic acid)

Synthesis of NDP-sugars• Two main pathways

1) Biosynthesis: The Interconversion PathwaySugar-->sugar-6-P-->sugar-1-P-->NDP-sugar(1)-->other NDP-sugars

Glc, Man, Frc

2) Catabolism: The Salvage PathwayPolysaccharide--->sugars-->sugars-1-P-->NDP-sugarsAlmost all other sugars in a cell

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The interconversion pathwayoverview

• Glc/ Man ---> Glc-6-P ---> Glc-1-P• Glc-1-P +UTP---> UDP-Glc• UDP-glucose-----> many different NDP-

sugars

ADP-α-D-Glc

Ara-5-P

GDP-β-L-Fuc GDP-4k-6d-D-ManGDP-α-D-Man

GDP-L-Gul

GDP-L-Gal

UDP-β-L-Rha

UDP-α-D-GlcUDP-α-D-Gal

UDP-4k-6d-D-Glcβ-L-Rha-1-PL-Rha

UDP-α-D-GlcA

UDP-α-D-GalA

UDP-α-D-Xyl UDP-D-Api

UDP-L-Arap

α-D-Glc-1-P

D-Frc-6-P

D-Glc-6-P D-Glc

Frc

D-Man-6-P D-Man

α-D-Man-1-P

GlcN-6-PGlcNac-1-PUDP-α-D-GlcNac

ADP-D-Gal

β-L-Fuc-1-PL-FucATP ADP GTP PPi

UTPPPi AcCoACoAGln

Glu

GTPPPi

Gal-1-PGlc-1-P

ATP

PPi

UTPPPi

ADPFrc

UDP

Frc

TDPFrcTDP-D-Glc

ADPATP PPiUTP

TDP-β−L-Rha

α-D-Gal-1-PADPATP PPiUTP

D-Gal

O2

α-D-GlcA-1-PADPATP PPiUTP

L-Ara-1-PADPATP PPiUTP

L-Ara

α-D-Xyl-1-PPPiUTP

Xyl

D-GlcA

α-D-GalA-1-PADPATP PPiUTP

D-GalAKDO-8-P

KDOCTP

CMP-KDO

PPi

UDP-α-D-sulfoQuin

R-SO3R

PEP

CO2

CO2

23

3

3

4

56

7

8

910

10

11 12 13

13

13

13 14

15

16

17

18 13

19 2013

1

21

2223

2425

262729 30

ATPADP

31

34

35

36

TDP-D-GlcA

4

GDP-D-Glc37GTP PPi

UDP-β-L-Araf

L-Gal-1-PL-Gal

38

3940

GDPPi40

41

43

42

Inositol

Ascorbate

ATPADP

32

ATPADP

33

NAD+

NADH

Sucrose

44

GlcGlc6P

Glc6P

Frc

GlcSucrose-6-P

4546

Frc6PUDP-Glc

Sucrose

inositol

Fructose

ER

Golgi

Glucose

PM

Glc6P

trehalsoe6P

Simple view of synthesis of activated-sugars in plantsBar-Peled, 2006

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The Interconversion Pathway• Converting Energy source: Glucose, Mannose, Fructose to Sug-6-P

Step 1.

6Highlyregulatedpathway

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Step 2: PGM“‘reversible”committed step to transform Sugar-6-P to Sugar-1-P

PhosphoglucoMutase(α-PGM)

Examples:GDP-Man

UDP-GlcTDP-GlcADP-Glc

Interconversion pathway cont.• NDP-sugar pyrophosphorylase (Ppase). A group of

reversible enzymes that transfer nucleotide (XMP)from XTP onto a sugar-1-P, forming NDP-sugar

Cardini, Leloir 1950 Cabib, Leloir 1953

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interconversion

• A nucleotide sugar is converted to anothernucleotide sugar by the action of differenttypes of enzyme activities:– 4,6-dehydratase– 6-oxidoreductase– Decarboxylase– 4, epimerases– 2-epirmrases– Mutarotase– 4-epimerase/reductase– 3,5-epimerase

Once UDP-Glc or GDP-Man are formed….

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Biosynthesis of UDP-Rha, UDP-GlcA, UDP-Xyl,UDP-Api, UDP-GalA,UDP-Ara

O

CH2OH

OH

OH

O-UDPOH

UDP-α-D-Glc

4,6-dehydrataseO

CH3

OH

OH

O-UDP

O

UDP-α-D-4keto-6deoxyGlc

O

OH

O-UDP

OH

CH3O

UDP-β-L-4keto-Rha

OOH

OH

O-UDP

OH

CH3

UDP-β-L-Rha

NAD+ 3,5-epimerase 4-keto reductase+ NADPHring flipNADH H2O

O-UDP

O

COOH

OH

OH

OH

UDP-α-D-GalA

4-epimerase

NAD+

NADHCO2

UDP-α-D-Api

O

OHOH

CH2OH

O-UDPUDP-Api syn

O-UDP

O

OH

OH

OH

UDP- α-D-Xyl

CO2decarboxylase

NAD+

NADH

UDP-α-D-GlcA

2NADH-H2O

2NAD+

O-UDP

O

COOH

OH

OH

OH

6-oxidoreductase,Dehydrogenase

4

O-UDP

O

OH

OH

OH

UDP- α-Arap

Enz

Enz*

UDP-GlcA

OHOOH

OHO- UDP

COO-

UDP-XylOHO

OHOH

O- UDP

NADHNAD+

NADH NAD+OH

OO

OHO-UDP

HCO2

O

OHOH

O-UDP

COO-

O+ H +H

H

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A plausible mechanism for the catalysis of the decarboxylationreaction. ArnA

Williams, G. J. et al. J. Biol. Chem. 2005;280:23000-23008

• A plausible mechanism for the catalysis of the decarboxylation reaction.The carboxylic acid group of substrate is shown protonated, although insolution we would have expected it to be deprotonated. However, theE434Q mutant is inactive, and we suggest that it is required to ensuredeprotonation of the keto intermediate. The carboxylate form of the ketointermediate is expected to spontaneously decompose without the needfor any further catalysis.

A decarboxylase

Structure, Vol. 13, 929–942, June, 2005

Petia,Structure, 2005. 13:929–942

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A 4-epimerase

SDR- related enzymes

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The Salvage Pathways• Releasing of a monosaccharide (sugar) from

storage carbohydrate/glycoprotein• Glycoprotein--> GlcNac

• Hydrolysis of sugars from polysaccharides• Cellulose--> Glc

• Releasing of sugar-1-P from storage• Glycogen--> Glc-1-P

• Converting a soluble disaccharide• Sucrose> UDP-Glc, Maltose-->Glc-1-P

Different species adopt different mechanism

examples

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Sugar-1-P kinase

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I,2. Special case Step A: Conversion of Galactoseto Gal1P; UDP-Glc to UDP-Gal

UDP-GlcNAc

PPAse

4-epimerase

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Not all sugar donor are NDP-sugars. isoprene phosphate-linked sugars are donor substrates for a

wide variety of glycosyltransferases

- GalA residues in the lipid A and core domains of R.leguminosarum LPS.

• In Escherichia coli, undecaprenyl diphosphate-sugars are substrates for the polymerization ofpeptidoglycan

• Undecaprenyl monophosphate-sugars are donors inthe biosynthesis of mycobacterial lipoglycans

• The structurally related dolichyl monophosphate- anddiphosphate-sugars of eukaryotic cells are requiredfor protein glycosylation

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Kanjilal-Kolar, S. et al. J. Biol. Chem. 2006;281:12879-12887

Proposed pathway and topography for attachment of GalA residues to the lipid A and coredomains of R. leguminosarum LPS

Proposed pathway and topography for attachment of GalA residues to the lipid A and core domains of R. leguminosarum LPS. Thesimplest scenario for the biosynthesis of dodecaprenyl-beta-D-GalA is as follows. 1 and 2, UDP-Glc is oxidized by the dehydrogenaseExo5 to UDP-GlcA, which is then converted by the C4-epimerase LpsL to UDP-GalA (22). 3, in analogy to PmrF (ArnC) (30) in E.coli, Orf3 transfers GalA from UDP-GalA to dodecaprenyl phosphate, generating dodecaprenyl phosphate-beta-D-GalA, which isflipped to the periplasmic leaflet by an unknown mechanism. As yet, we have not been able to assay Orf3 in vitro. 4, lipid A and coreLPS sugars are synthesized on the cytoplasmic leaflet of the inner membrane (43-46, 56, 57) and flipped to the periplasmic leaflet byMsbA. 5 and 6, on the periplasmic side of the inner membrane the 1- and 4'-phosphatases, LpxE and LpxF (40, 50), dephosphorylatelipid A, creating the substrate for GalA addition. 7-10, GalA is transferred from dodecaprenyl-beta-D-GalA to the outer Kdo by RgtAand RgtB, and to mannose by RgtC.

Kanjilal-Kolar, S. et al. J. Biol. Chem. 2006;281:12879-12887

ESI/MS/MS analysis of the putative GalA donor substrate for RgtA

Biosynthesis of isoprenoid;isoprenyl diphosphate synthase

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A UDP-MurNAc-pentapeptide (Park Nucleotide is a donor ) forUndecaprenyl diphosphate-MurNAc-pentapeptide-GlcNAc

Raetz Anal Biochem 2005

Park

nucleotide

Samuelson, John et al. (2005) Proc. Natl. Acad. Sci. USA 102, 1548-1553

Fig. 1. The inventory of Alg glycosyltransferases and predicted dolichol-linked glycans varydramatically among protists and fungi

These glycosyltransferases add phospho-GlcNAc to dolichol phosphate,in the cytosol

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Do nucleotide-sugars control synthesis ofpolysaccharides in the cell?

How alteration in the balance; flux of NDP-sugarsmodified glycan synthesis

Why in evolution the same sugar (e.g. glucose) boundto different nucleotides (ADP, GDP, UDP, TDP)?

What mechanism control glycan synthesis? NDP-sugars or Glycosyltransferases?

HOT Questions;Precursors synthesis and regulation

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Precursors transport across membranes and regulation