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J. Biol. Chem., Vol. 279, Issue 24, 25789-25797, June 11, 2004

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

Suzanne C. Edavettal, Karen A. Lee, Masahiko Negishi¶, Robert J. Linhardt||, Jian Liu**, and Lars C. Pedersen

From the Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599, Laboratory of Structural Biology and ^¶Laboratory of Reproductive and Developmental Toxicology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, and ^||Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, New York 12180

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 (SO₃) 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-O-sulfotransferase 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.

Received for publication, January 30, 2004 , and in revised form, March 10, 2004.

The atomic coordinates and structure factors (code 1S6T) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported in part by National Institutes of Health Grant AI50050 (to J. L.) and American Heart Association Mid-Atlantic Affiliate Grant-in-aid 0355800U (to J. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence and reprint requests should be addressed: Rm. 309, Beard Hall, University of North Carolina, Chapel Hill, NC 27599. Tel: 919-843-6511; Fax: 919-843-5432; E-mail: jian_liu{at}unc.edu.

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