Edavettal Suzanne C, Lee Karen A, Negishi Masahiko, Linhardt Robert J, Liu Jian, Pedersen Lars C
Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
J Biol Chem. 2004 Jun 11;279(24):25789-97. doi: 10.1074/jbc.M401089200. Epub 2004 Apr 1.
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-O-sulfotransferase isoform 1 at 2.5-A 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.
硫酸乙酰肝素与抗凝血酶（一种蛋白酶抑制剂）相互作用，以调节血液凝固。硫酸乙酰肝素3-O-磺基转移酶同工型1在抗凝硫酸乙酰肝素的生物合成中执行关键的最后一步修饰。该酶将硫酸基（SO(3)）从3'-磷酸腺苷5'-磷酸硫酸转移到氨基葡萄糖残基的3-OH位置，形成3-O-磺基氨基葡萄糖，这是硫酸乙酰肝素与抗凝血酶结合的关键结构基序。在本研究中，我们报道了3-O-磺基转移酶同工型1与3'-磷酸腺苷5'-磷酸形成的二元复合物在2.5埃分辨率下的晶体结构。该结构揭示了对3'-磷酸腺苷5'-磷酸硫酸结合至关重要的残基，并暗示了硫酸乙酰肝素结合所需的残基。此外，定点诱变分析表明，精氨酸-67、赖氨酸-68、精氨酸-72、谷氨酸-90、组氨酸-92、天冬氨酸-95、赖氨酸-123和精氨酸-276残基对酶活性至关重要。在这些必需氨基酸残基中，我们发现精氨酸-67、精氨酸-72、组氨酸-92和天冬氨酸-95残基在硫酸乙酰肝素3-O-磺基转移酶中保守，但在硫酸乙酰肝素N-脱乙酰酶/N-磺基转移酶中不保守，表明这些残基在赋予底物特异性方面发挥作用。本研究结果为理解抗凝硫酸乙酰肝素的生物合成以及硫酸乙酰肝素磺基转移酶的一般作用机制提供了重要信息。
