Glycobiology 8130, 18/1/2005 Spring 2005

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1 Glycobiology 8130, 18/1/2005 Spring 2005 Nucleotide-sugar donors. Maor Bar-Peled, CCRC; Plant Biology ISTRY 1906 arden and Young- discovered that yeast fermented with sugar accumulate of phosphate esters-sugars. Using the methods they established, the chemistry, synthesis, and the metabolic roles of sugar-phosphates were start to emerge. ehre 1951, was first to observed that an enzyme catalyze the transfer of specific sugar form (sugar-phosphate donor) onto another sugar acceptor 1

2 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 ) GlyT display remarkable diversity in their donor, acceptor and product specificity and generate a potentially infinite number of glycoconjugates, oligo- and polysaccharides. --Survey of glycosyltransferase-related sequences in the database reveal over 7200 GT s to date. Sugar donor Activated-Sugar + Acceptor After Edelman, 1956 GlyT sugar-acceptor The diversity of glycan is contributed, in part, by the number of sugar-donors each organism is making Role of a nucleotide sugars Yeast, human: 8-12 nucleotide-sugars Plants: >30 Prokaryotes: >70 Nucleotide-sugars are precursors for synthesis of macromolecules: Glycoprotein, Glycolipids, Proteoglycans. ligosaccharides; Polysaccharides. Nucleotide-sugars are precursors for synthesis of small molecules: Toxins, Antibiotics, Secondary metabolites, ormones, etc.. 2

3 1950- the discovery of nucleotide-sugars. Sugar-donor for glycan synthesis Biosynthesis of glycan: A specific GT attach a sugar from NDP-sugars 3

4 Biosynthesis of activated sugar donors Three major routs: I- sugar activation 1- Sugar-6-phosphate isomerization (Glycolysis: Glc-6-P, Man-6-P) 2- Sugar-1-P kinases (Gal1P, GlcA1P, Fuc1P, etc..) II-nucleotide-sugar interconversion III-CMP-sugars 1- dolichol-p[p]-sugar 2- myo-inositol-galactinol ther activated-sugars Figure 6.1. Biosynthesis and interconversion of monosaccharides. The relative contributions of each under physiological conditions are unknown. (Shadowed rectangles and shadowed ovals) Donors; (open ovals) monosaccharides; (stars) control points. From Glycobiology 4

5 I. 1. First route: synthesis of sugar-6-p hexokinase Phospho-mutase Sugar ATP Sugar-6-P Sugar 1-P Glucose Mannose GlcNac ighly regulated pathway. I.1 Step A: glucokinase (K) Glc-6-P Glc+ ATP+Mg----Glc-6-P +ADP++ The nucleophilic attack of the C6- group of glucose on the phosphate of an Mg2+-ATP complex. The Mg2+ functions to shield the negatively charged groups of ATP, thus facilities the nucleophilic attack + 5

6 I.1 Step B: Phosphoglucose mutase Cardini,Paladini, 1949 Man-1-P exokinase, converts mannose with ATP to Man-6-P, and phosphomannose isomerase converts Man6P to Fru6P (aldose to ketose) similar to phosphglucose isomerase I.1,2 Step C: PPases. Conversion of sugar- 1P to NDP-sugars Glc-1-P + UTP UDP-Glc + PPi 2Pi Man-1-P + GTP GDP-Man + PPi 2Pi Cardini, Leloir 1950 Cabib, Leloir

7 I.1,2 C: Synthesis of UDP-Glc UDP-Glucose pyrophosphorylase. The phosphoryl oxygen of G1P attacks the alpha phosphous atom of UTP to form UDP- Glc and PPi. The PPi is rapidly hydrolyze with water by inorganic pyrophosphatse 2 I, 2: direct sugar-1p- kinase Kinase PPase NTP PPi sugar ATP Sugar-1-P NDP-Sugar Arabinose Glucuronic acid Rhamnose Xylose GalNac Fucose Galactose Salvage pathway? Caputto, Leloir, The specificities of the kinase to the various sugars is not clear yet. -The specificities of the pyrophosphorylase (PPases) is also unclear. 7

8 I,2. Special case Step A: Conversion of Galactose to Gal1P; UDP-Glc to UDP-Gal Caputto,Leloir 1950 Cardini, Leloir Galactokinase, Gal is phosphorylated at C1 by ATP. 2. Gal-1-P uridylyltransferasetransferase transfers UDP-Glc s urydylyl group to Gal-1-P to yield Glc-1-P and UDP-Gal by the reversible cleavage of UDP-Glc s pyrophosphoryl bond. 3. UDP-Gal Epimerase. II: nucleotide-sugar interconversion route C 3 -UDP UDP-4K-L-Rha C3 -UDP UDP-L-Rha UDP-Rha Epi/Red NRSER C 3 C 2 -UDP UDP-D-Api UDP-Rha Dehydratase -UDP UDP-4K-6d-D-Glc C C 2 C 2 -UDP UDP-D-Glc 2NAD UDP-Glc 2NAD Dehydrogenase UGD C -UDP UDP-D-GlcA 3 UDP-GlcA Decraboxylase UXS 2 UDP-Glc Epimerase 4 UDP-GlcA epimerase C 2 -UDP UDP-D-Gal C -UDP UDP-D-GalA Interconversion of UDP-sugars in plants -UDP UDP-D-Xyl 6 UDP-Xyl epimerase -UDP UDP-L-Aral 8

9 UDP-galactose-4-epimerase A reversible 4-epimerase. Converts UDP-Galactose to UDP- Glucose. This enzyme has an associated NAD+, indicates that the reaction involves the sequential oxidation and reduction of the hexose C4 atom Structure of reduced form of NAD and NADP and their oxidized form NAD and NADP Synthesis of GDP-Fuc from GDP-Man 9

10 Route 3 III. Fusion of CMP to 7-9 carbon sugars Sugar (7-9 carbon) CTP PPi CMP-Sugar KD Neu5AC[Sialic acid (NANA)] Proposed Pathway for the Assembly of Arabinogalactan, its Attachment to Peptidoglycan and Subsequent Mycolyation. Glc-1-P rmla UDP-GlcNAc P-polyprenyl TDP-Glc rmlb TDP-4-keto-6deoxyGlc rmlc TDP-4-keto-Rha rmld UMP rfe GlcNAc-P-P-polyprenyl TDP-Rha TDP wbbl 30 UDP-Glc Rha- GlcNAc-P-P-polyprenyl gale 30 UDP-Galp 30 UDP-Galf glf Glactofuranosyl Transferases I, II & III 30 UDP 70 Rib-5-P Galf30-Rha- GlcNAc-P-P-polyprenyl 70 prpp 70 DPA DPA Formation Enzymes 70 DP ~ 480 Acetate 16 Myc PL? Mycolyl Formation Enzymes Multiple Arabinofuranosyl Transferases Araf70-Galf30- Rha- GlcNAc-P-P-polyprenyl Mycolyl Transferases Naismith, PL Myco16-Araf70-Galf30- Rha- GlcNAc-P-P-polyprenyl + Peptidoglycan Structural Biology P-polyprenyl Centre mag Transferase for Biomolecular Sciences The University, St. Andrews 10

11 dttp RmlA The rhamnose pathway P 3 PPi dtdp-rhamnose immediate precursor of rhamnose Made in four steps RmlA glucose-1_phosphate thymidyltransferase (EC ) RmlB dtdp-d-glucose 4,6-dehydratase (EC ) RmlC dtdp-6-deoxy-d-xylo-4-hexulose 3,5-epimerase (EC ) RmlD dtdp-6-deoxy-l-lyxo-4-hexulose reductase (EC ) -dtdp (NAD+) -2 RmlB C3 RmlC -dtdp C3 -dtdp ring flip (non-enzymatic) 3C NADP RmlD -dtdp + NADP 3C Structural Biology -dtdp Centre for Biomolecular Sciences Frey; The Naismith, 2001 University, St. Andrews NAD+ RmlB NAD- oxidation (hydride abstraction) TDP TDP-glucose NAD glucosene NAD- TDP proton abstraction TDP TDP dehydration S. Typhimurium solved by mol replacement (Coli Thoden/olden) TDP NAD+ reduction TDP dtdp-4-keto 6-deoxy-D-glucose Structural Biology Centre for Biomolecular Sciences The University, St. Andrews 11

12 dtdp-glucose bound to S. suis (left) & S. typhimurium Structural Biology Centre for Biomolecular Sciences The University, St. Andrews Ligand binding changes structure C-terminus orders and covers active site Mainchain at active site moves, key residues at active site perturbed Also seen with dtdp Potentiates NAD Structural Biology Centre for Biomolecular Sciences The University, St. Andrews 12

13 * Key contacts NAD * Structural Biology Centre for Biomolecular Sciences The University, St. Andrews * Triad marked with *, Glu 135 and Asp 134 conserved in all dehydratases Tyr 167 acts as principal base (2.6A) Thr 133 regulates pka of 4 Lys 171 stabilise Tyr 167 C4 distance TDPG to C4 NAD is 3.2A Glu 135 perfectly position to remove from C5 position 6 strong -bond to Asp 134 (water bond to 134 in xylose) Some special Activated sugars In plant chloroplast and bacteria : UDP-Glc is converted to UDP-6-sufo-derivative 13

14 UDP-GlcNac UDP-Glc GDP-Man Synthesis of N-linked glycoprotein in ER- is mediated by dolichol-activated sugars GDP -Pcytosol Man -P- flip -P- Man ER-lumen SUGAR Dolichol pyrophosphate glycoside are the carbohydrate precursors of N-linked glycosides. The sugar units to be added are chemically activated by attachment to isoprenoid alcohols called dolichols-pyrophosphate or mono-p [ /\/\/\/\_P]. These molecule are anchored by hydrophobic interactions with membrane lipids. Synthesis of NDP-sugars is both in the cytosol and in organelles 14

15 Transporters for sugar nucleotides, PAPS, and ATP are located in the Golgi membranes of mammals, yeast, protozoa, and plants. These proteins are actually antiporters, and the corresponding nucleoside monophosphate is carried into the cytosol with sugar nucleotide transport. Since most glycosylation reactions produce a nucleoside diphosphate, this requires conversion to the nucleoside monophosphate. For PAPS, the corresponding exiting molecule is unknown, and for ATP, it is AMP, ADP, or both. The phosphate (Pi) transporter is hypothetical. From Glycobiology Biosynthesis, utilization, and turnover of a common monosaccharide. This schematic shows the biosynthesis, fate, and turnover of one common monosaccharide constituent of animal glycans, galactose. Although small amounts of galactose can be taken up from the outside of the cell, most is either synthesized de novo from glucose or recycled from degradation of glycoconjugates in the lysosome. The schematic presents a simplified view of the generation of the UDP sugar nucleotide, its equilibrium state with UDP-glucose, and its uptake and utilization in the Golgi apparatus for synthesis of new glycans. (Solid lines) Biochemical pathways; (dashed lines) pathways for the trafficking of membranes and glycans. 15

16 Tunicamycin: Inhibition of DL-PP-GlcNAc AssemblyFigure Structure of tunicamycin. Tunicamycin consists of uridine conjugated to the dialdose, tunicamine. The compound is part of a family of related agents that vary in the length, branching, and degree of unsaturation of the fatty acid amide (R) linked to tunicamine. Mutant in the Synthesis of NDP-sugars UDP-Glc-dehydrogenase: eart valve defect UDP-Galactose: Galactosemia GDP-Fuc: 16

17 GALACTSEMIA Figure UDP-Gal synthesis and galactosemia. The most common form of galactosemia is due to a deficiency of Gal-1-P uridyltransferase (GalT). This enzyme normally utilizes Gal-1-P derived from dietary galactose. In the absence of GALT, Gal-1-P accumulates, along with excessive galactose and its oxidative and reductive products galactitol and galactonate (not shown). UDP-Gal synthesis may also be impaired in the absence of GALT, but not completely so, since UDP-Gal 4-epimerase (GALE) can form UDP-Gal from UDP- Glc, and can supply the galactosyltransferases required for normal glycoconjugate biosynthesis. 17