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Glycobiology 8130,
Lecture 2
8/1/2007
NDP-sugar synthesis
Maor Bar-Peled
CCRC
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
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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?