1

Glycobiology 8130,

Lecture 2

8/1/2007

NDP-sugar synthesis

Maor Bar-Peled

CCRC

Peled@ccrc.uga.edu

History

• ~100 years ago (1906) Harden and Yo

discovered that yeast fermented with sugar accumulate aphosphate esters-sugar derivative. Man-6-P, Glc-6-P

! Using the methods they established, the synthesis, andthe metabolic roles of sugar-phosphates were start toemerge.

!The synthesis of a polysaccharide from glucose-1-phosphate inmuscle extract. Carl F. Cori, Gerhard Schmidt, and Gerty T. CoriScience 19 May 1939

1947 Nobel Prize. Cori!s

(Glycogen synthesis, metabolism& disease)

EMIL FISCHER observed in 1890 that L-glucose was not metabolized

by brewer' yeast

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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 his early collaborators, Ranwel Caputto,

Carlos E. Cardini, Raúl Trucco and Alejandro C.

Paladini work was started on the metabolism of

galactose which led to the isolation of glucose

1,6-diphosphate and uridine diphosphate

glucose.

UDP-gluocse served as

glucose donor in the

synthesis of trehalose

(with Enrico Cabib, 1953 )

and sucrose (with Carlos

E. Cardini and

J.Chiriboga, 1955) 1970, Nobel Proze

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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

Sugar Nucleotide

PO

O

P

O

O

O-

1

23

4

O

OH

HO

5

HO

CH2OH

O CH25! O

OHOH

HN

O

O N1

2

3 4 5

6

2!3!

4!

u

rib

O-

A Glycosyl-transferase uses a nucleotide-

sugars to build a glycan

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

catalyze the transfer of a sugar moiety from an activated sugar donor onto

saccharide or non-saccharide acceptors. !

• GlyT attaches of the sugar in a specific linkage (alpha, beta 1-2 1-4) ! GlyT display remarkable diversity in their donor, acceptor and productspecificity and generate a potentially infinite number of glycoconjugates, oligo- and

polysaccharides. !

--Survey ofglycosyltransferase-related sequences inthe database revealover 7200 GTs todate.

Most but not all -areusing NDP-sugar asdonors

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,

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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/ Recycling: Salvage Pathwaysugars-->sugars-1-P-->NDP-sugars

Almost all other sugars in a cell

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

Step 1.

6Highly

regulated

pathway

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

PhosphoglucoMutase (!-PGM)

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Examples:

GDP-Man

UDP-Glc

TDP-Glc

ADP-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….

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

O

CH2OH

OH

OH

O-UDPOH

UDP-!-D-Glc

4,6-dehydrataseO

CH3

OH

OH

O-UDP

O

UDP-!-D-

4keto-6deoxyGlc

O

OH

O-UDP

OH

CH3O

UDP-"-L-

4keto-Rha

OOH

OH

O-UDP

OH

CH3

UDP-"-L-Rha

NAD+ 3,5-epimerase 4-keto reductase

+ NADPHring flipNADH H2O

O-UDP

O

COOH

OH

OH

OH

UDP-!-D-GalA

4-epimerase

NAD+

NADHCO2

UDP-!-D-Api

O

OHOH

CH2OH

O-UDP

UDP-Api syn

O-UDP

O

OH

OH

OH

UDP- !-D-Xyl

CO2decarb

oxylas

e

NAD+

NADH

UDP-!-D-GlcA

2NADH

-H2O

2NAD+

O-UDP

O

COOH

OH

OH

OH

6-oxidoreductase,

Dehydrogenase

4

O-UDP

O

OH

OH

OH

UDP- !-Arap

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Enz

Enz*

UDP-GlcA

OHO

OHOH

O- UDP

COO-

UDP-Xyl

OHO

OHOH

O- UDP

NADHNAD+

NADH NAD+OH

OO

OHO-UDP

HCO2

O

OHOH

O-UDP

COO-

O+ H +

A plausible mechanism for the catalysis of the decarboxylation

reaction. 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 in

solution we would have expected it to be deprotonated. However, the

E434Q mutant is inactive, and we suggest that it is required to ensure

deprotonation of the keto intermediate. The carboxylate form of the keto

intermediate is expected to spontaneously decompose without the need

for any further catalysis.

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Salvage / Recycling 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 di-saccharide• 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 Galactose

to Gal1P; UDP-Glc to UDP-Gal

GalT-Not in plants

UDP-GlcNAc

PPAse

4-epimerase

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Not all sugar donor are NDP-sugars.

isoprene phosphate-linked sugars are donor substrates for awide 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 core

domains of R. leguminosarum LPS

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

simplest scenario for the biosynthesis of dodecaprenyl-beta-D-GalA is as follows. 1 and 2, UDP-Glc is oxidized by the dehydrogenase

Exo5 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 is

flipped to the periplasmic leaflet by an unknown mechanism. As yet, we have not been able to assay Orf3 in vitro. 4, lipid A and core

LPS sugars are synthesized on the cytoplasmic leaflet of the inner membrane (43-46, 56, 57) and flipped to the periplasmic leaflet by

MsbA. 5 and 6, on the periplasmic side of the inner membrane the 1- and 4'-phosphatases, LpxE and LpxF (40, 50), dephosphorylate

lipid A, creating the substrate for GalA addition. 7-10, GalA is transferred from dodecaprenyl-beta-D-GalA to the outer Kdo by RgtA

and 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 ) for

Undecaprenyl diphosphate-MurNAc-pentapeptide-GlcNAc

Raetz Anal Biochem 2005

Park

nucleotid

e

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 of

polysaccharides in the cell?

How alteration in the balance; flux of NDP-sugars

modified glycan synthesis

Why in evolution the same sugar (e.g. glucose) bound

to different nucleotides (ADP, GDP, UDP, TDP)?

What mechanism control glycan synthesis? NDP-

sugars or Glycosyltransferases?