Expression in Escherichia coli, Purification and Kinetic Characterization of Human Heparan Sulfate 3-O-Sulfotransferase-1

Myette, James R.; Shriver, Zachary; Liu, Jian; Venkataraman, Ganesh; Rosenberg, Robert D.; Sasisekharan, Ram

doi:10.1006/bbrc.2001.6268

Cited by 11 publications

(4 citation statements)

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“…The dissociation constant of PAP with wild-type 3-OST-1 has been determined by isothermal titration calorimetry (ITC) to be 14 ( 4 µM (27). This value is similar to the K m value of 10 µM for PAPS with 3-OST-1 (33), suggesting that the PAP represents a good model for PAPS in studying the binding affinity to 3-OST-1. To measure the interaction of the binary complex 3-OST-1-PAP with HS, 3-OST-1 in phosphate running buffer containing an excess amount of PAP (40 µM) was injected over an HS sensor chip (FC4) at different concentrations (250-2000 nM).…”

Section: T H I S C O N T E N T Isupporting

confidence: 78%

Affinity, Kinetic, and Structural Study of the Interaction of 3-O-Sulfotransferase Isoform 1 with Heparan Sulfate

Muñoz

Kemp

et al. 2006

Biochemistry

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The 3-O-sulfonation of glucosamine residues in heparan sulfate (HS) by 3-O-sulfotransferase (3-OST) is a key substitution that is present in HS sequences of biological importance, in particular HS anticoagulant activity. Six different isoforms of 3-OST have been identified that exhibit different substrate specificity. In this paper the affinity and kinetics of the interaction between 3-O-sulfotransferase isoform 1 (3-OST-1) and HS have been examined using surface plasmon resonance (SPR). 3-OST-1 binds with micomolar affinity to HS (K(D) = 2.79 microM), and this interaction is apparently independent of the presence of the coenzyme, 3'-phosphoadenosine 5'-phosphosulfate (PAPS). A conformational change in the complex has also been detected, supporting data from previous studies. Selected 3-OST-1 mutants have provided valuable information of amino acid residues that participate in 3-OST-1 interaction with HS substrate and its catalytic activity. The results from this study contribute to understanding the substrate specificity among the 3-OST isoforms and in the mechanism of 3-OST-1-catalyzed biosynthesis of anticoagulant HS.

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Section: T H I S C O N T E N T Isupporting

confidence: 78%

Affinity, Kinetic, and Structural Study of the Interaction of 3-O-Sulfotransferase Isoform 1 with Heparan Sulfate

Muñoz

Kemp

et al. 2006

Biochemistry

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“…The use of PAP as an analog for mimicking the interaction between PAPS and sulfotransferases is a widely used approach, provided that the binding affinity of sulfotransferases to PAP is similar to the affinity of sulfotransferase to PAPS (18). Indeed, we determined the K D of 3-OST-1 (WT) for PAP to be 14 M, very close to the 10 M K m of wild type 3-OST-1 for PAPS, determined by kinetic analysis (35). Our result suggests that the PAP binding affinity of 3-OST-1 provides a good estimate for the binding affinity to PAPS.…”

Section: Resultsmentioning

confidence: 72%

Crystal Structure and Mutational Analysis of Heparan Sulfate 3-O-Sulfotransferase Isoform 1

Edavettal

Lee

Negishi

et al. 2004

Journal of Biological Chemistry

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Heparan sulfate interacts with antithrombin, a protease inhibitor, to regulate blood coagulation. Heparan sulfate 3-O-sulfotransferase isoform 1 performs the crucial last step modification in the biosynthesis of anticoagulant heparan sulfate. This enzyme transfers the sulfuryl group (SO 3 ) from 3-phosphoadenosine 5-phosphosulfate to the 3-OH position of a glucosamine residue to form the 3-O-sulfo glucosamine, a structural motif critical for binding of heparan sulfate to antithrombin. In this study, we report the crystal structure of 3-Osulfotransferase isoform 1 at 2.5-Å resolution in a binary complex with 3-phosphoadenosine 5-phosphate. This structure reveals residues critical for 3-phosphoadenosine 5-phosphosulfate binding and suggests residues required for the binding of heparan sulfate. In addition, site-directed mutagenesis analyses suggest that residues Arg-67, Lys-68, Arg-72, Glu-90, His-92, Asp-95, Lys-123, and Arg-276 are essential for enzymatic activity. Among these essential amino acid residues, we find that residues Arg-67, Arg-72, His-92, and Asp-95 are conserved in heparan sulfate 3-O-sulfotransferases but not in heparan N-deacetylase/N-sulfotransferase, suggesting a role for these residues in conferring substrate specificity. Results from this study provide information essential for understanding the biosynthesis of anticoagulant heparan sulfate and the general mechanism of action of heparan sulfate sulfotransferases.

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“…This is followed by sulfation at the C6 position by two isoforms of 6- O -sulfotransferase (6-OST-1 & −3) in the presence of the cofactor regeneration system leading to generation of non-anticoagulant heparin structure (Chen et al, 2005; Restaino et al, 2013; Zhang et al, 2001; Zhang et al, 2008). 3- O -sulfotransferase-1 (3-OST-1) then sulfates the C3 position, also in the presence of the cofactor regeneration system to generate anticoagulant heparin (Myette et al, 2002; Zhang et al, 2008). A similar sequential approach led to another version of bioengineered heparin derived from partially N -deacteylated/ N -sulfonated heparosan as substrate (Wang et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

Combinatorial one-pot chemoenzymatic synthesis of heparin

Bhaskar

et al. 2015

Carbohydrate Polymers

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Contamination in heparin batches during early 2008 has resulted in a significant effort to develop a safer bioengineered heparin using bacterial capsular polysaccharide heparosan and recombinant enzymes derived from the heparin/heparan sulfate biosynthetic pathway. This requires controlled chemical N-deacetylation/ N-sulfonation of heparosan followed by epimerization of most of its glucuronic acid residues to iduronic acid and O-sulfation of the C2 position of iduronic acid and the C3 and C6 positions of the glucosamine residues. A combinatorial study of multi-enzyme, one-pot, in vitro biocatalytic synthesis, carried out in tandem with sensitive analytical techniques, reveals controlled structural changes leading to heparin products similar to animal- derived heparin active pharmaceutical ingredients. Liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy analysis confirms an abundance of heparin’s characteristic trisulfated disaccharide, as well as 3-O-sulfo containing residues critical for heparin binding to antithrombin III and its anticoagulant activity. The bioengineered heparins prepared using this simplified one-pot chemoenzymatic synthesis also show in vitro anticoagulant activity.

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Expression in Escherichia coli, Purification and Kinetic Characterization of Human Heparan Sulfate 3-O-Sulfotransferase-1

Cited by 11 publications

References 50 publications

Affinity, Kinetic, and Structural Study of the Interaction of 3-O-Sulfotransferase Isoform 1 with Heparan Sulfate

Affinity, Kinetic, and Structural Study of the Interaction of 3-O-Sulfotransferase Isoform 1 with Heparan Sulfate

Crystal Structure and Mutational Analysis of Heparan Sulfate 3-O-Sulfotransferase Isoform 1

Combinatorial one-pot chemoenzymatic synthesis of heparin

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