Molecular Mechanism of Substrate Specificity for Heparan Sulfate 2-O-Sulfotransferase

Liu, Chunhui; Sheng, Juzheng; Krahn, J.M.; Perera, Lalith; Xu, Yongmei; Hsieh, Po Hung; Dou, Wenfang; Liu, Jian; Pedersen, Lars C.

doi:10.1074/jbc.m113.530535

Cited by 45 publications

(82 citation statements)

References 38 publications

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“…1B). The NDST modification step is essential for the biosynthesis of HS as other HS biosynthetic enzymes, such as C 5 -epimerase (6, 7), 2-O-sulfotransferase (8,9), and 3-O-sulfotransferase (10), all require the presence of GlcNS residues to complete their modifications (Fig. 1A).…”

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confidence: 99%

Role of Deacetylase Activity of N-Deacetylase/N-Sulfotransferase 1 in Forming N-Sulfated Domain in Heparan Sulfate

Dou

Pagadala

et al. 2015

Journal of Biological Chemistry

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Background: N-Deacetylase/N-Sulfotransferase is a bifunctional enzyme in the heparan sulfate biosynthetic pathway. Results: N-Deacetylase and N-sulfotransferase domains display cooperative effects as a bifunctional enzyme; individually expressed N-deacetylase and N-sulfotransferase domains do not exhibit the same level of cooperativity. Conclusion:The bifunctionality of N-deacetylase/N-sulfotransferase is required to form N-S domain in heparan sulfate. Significance: This work uncovers the process by which NDST-1 constructs functional heparan sulfate.

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confidence: 99%

Role of Deacetylase Activity of N-Deacetylase/N-Sulfotransferase 1 in Forming N-Sulfated Domain in Heparan Sulfate

Dou

Pagadala

et al. 2015

Journal of Biological Chemistry

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“…For example, synthetic heparin oligosaccharides prepared by chemoenzymatic synthesis were used to solve the co-crystal structure of 3-OST-1/PAP/heptasaccharide and 2-OST/heptasaccharides/3′-phosphoadenosine-5′-phosphate. 44,53 These crystal structures helped us understand how sulfotransferases distinguish the specific saccharide sequences to exhibit substrate specificity. The results from these studies will, in turn, further improve oligosaccharide the synthesis using these enzymes.…”

Section: Discussionmentioning

confidence: 99%

“…Likewise, substituting the N-sulfo group with an N-acetyl group or hydrogen on the GlcN residue of the substrate abolishes its reactivity to 2-OST modification. 44 In contrast, two amino acid residues, Tyr-173 and Pro-82 near that active site of 2-OST are probably used by the enzyme to exclude the 6-O-sulfo group, suggesting that 6-O-sulfation occurs after 2-O-sulfation in the process of biosynthesizing highly sulfated domains consisting of -IdoA2S-GlcNS6S-, commonly found in heparin. The structural and biochemical evidence clearly demonstrates the essential roles of N-sulfation and 6-O-sulfation in regulating the 2-O-sulfation in the HS biosynthetic pathway.…”

Section: Design Of the Sequence Of Enzymatic Modificationsmentioning

confidence: 99%

“…Such a feature assures the biosynthesis of HS follows a preprogrammed modification sequence. 44 For example, 2-O-sulfation step catalyzed by 2-OST occurs only after N-sulfation step, but prior to the 6-O-sulfation step. The ternary crystal structure of 2-OST reveals that three amino acid residues, Arg-80, Lys-350 and Arg-190, from 2-OST directly interact with the N-sulfo group of GlcNS residue flanked on both end of the IdoA residue.…”

Section: Design Of the Sequence Of Enzymatic Modificationsmentioning

confidence: 99%

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Chemoenzymatic synthesis of heparan sulfate and heparin

Zhou

O’Leary

et al. 2012

Biocatalysis and Biotransformation

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Heparan sulfate is a polysaccharide that plays essential physiological functions in the animal kingdom. Heparin, a highly sulfated form of heparan sulfate, is a widely prescribed anticoagulant drug worldwide. The heparan sulfate and heparin isolated from natural sources are highly heterogeneous mixtures differing in polysaccharide chain lengths and sulfation patterns. The access to structurally defined heparan sulfate and heparin is critically important to probe the contribution of specific sulfated saccharide structures to biological functions as well as for the development of the next generation of heparin-based anticoagulant drugs. The synthesis of heparan sulfate and heparin, using a purely chemical approach, has proven extremely difficult, especially for targets larger than octasaccharides having a high degree of site-specific sulfation. A new chemoenzymatic method has emerged as an effective alternative approach. This method utilizes recombinant heparan sulfate biosynthetic enzymes combined with unnatural uridine diphosphate-monosaccharide donors. Recent examples demonstrate the successful synthesis of ultra-low molecular weight heparin, low-molecular weight heparin and bioengineered heparin with unprecedented efficiency. The new method opens the opportunity to develop improved heparinbased therapeutics.

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“…KfoC (GenBank TM accession number AB079602) was recombinantly expressed in E. coli BL21 (DE3) cells as a soluble N-His6-tagged fusion protein and purified using an appropriate affinity chromatography system. 4 Preparation of Model Oligosaccharide Substrates-Four structurally defined oligosaccharides were prepared using enzymatic synthesis following previously published procedures from the literature (32,33,35). All four of the oligosaccharides prepared in the current study were synthesized from the commercially available monosaccharide of GlcA-pNP.…”

Section: Methodsmentioning

confidence: 99%

Uncovering the Catalytic Direction of Chondroitin AC Exolyase

Yin

Wang

Sheng

2016

Journal of Biological Chemistry

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Glycosaminoglycans (GAGs) are polysaccharides that play vital functional roles in numerous biological processes, and compounds belonging to this class have been implicated in a wide variety of diseases. Chondroitin AC lyase (ChnAC) (EC 4.2.2.5) catalyzes the degradation of various GAGs, including chondroitin sulfate and hyaluronic acid, to give the corresponding disaccharides containing an ⌬ 4 -unsaturated uronic acid at their non-reducing terminus. ChnAC has been isolated from various bacteria and utilized as an enzymatic tool for study and evaluating the sequencing of GAGs. Despite its substrate specificity and the fact that its crystal structure has been determined to a high resolution, the direction in which ChnAC catalyzes the cleavage of oligosaccharides remain unclear. Herein, we have determined the structural cues of substrate depolymerization and the cleavage direction of ChnAC using model substrates and recombinant ChnAC protein. Several structurally defined oligosaccharides were synthesized using a chemoenzymatic approach and subsequently cleaved using ChnAC. The degradation products resulting from this process were determined by mass spectrometry. The results revealed that ChnAC cleaved the ␤1,4-glycosidic linkages between glucuronic acid and glucosamine units when these bonds were located on the reducing end of the oligosaccharide. In contrast, the presence of a GlcNAc-␣-1,4-GlcA unit at the reducing end of the oligosaccharide prevented ChnAC from cleaving the GalNAc-␤1,4-GlcA moiety located in the middle or at the non-reducing end of the chain. These interesting results therefore provide direct proof that ChnAC cleaves oligosaccharide substrates from their reducing end toward their non-reducing end. This conclusion will therefore enhance our collective understanding of the mode of action of ChnAC.

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Molecular Mechanism of Substrate Specificity for Heparan Sulfate 2-O-Sulfotransferase

Cited by 45 publications

References 38 publications

Role of Deacetylase Activity of N-Deacetylase/N-Sulfotransferase 1 in Forming N-Sulfated Domain in Heparan Sulfate

Role of Deacetylase Activity of N-Deacetylase/N-Sulfotransferase 1 in Forming N-Sulfated Domain in Heparan Sulfate

Chemoenzymatic synthesis of heparan sulfate and heparin

Uncovering the Catalytic Direction of Chondroitin AC Exolyase

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