Crystal structure and mutational analysis of heparan sulfate 3-O-sulfotransferase isoform 1
Crystal structure and mutational analysis of heparan sulfate 3-O-sulfotransferase isoform 1
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DOI：
10.1074/jbc.m401089200
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发表时间：
2004-06-11
影响因子：
4.8
通讯作者：
Pedersen, LC
中科院分区：
文献类型：
--
作者：
Edavettal, SC;Lee, KA;Pedersen, LC
硫酸肝素与抗凝血酶（一种蛋白酶抑制剂）相互作用，调节血液凝固。硫酸肝素3- o -硫转移酶异构体1在抗凝血剂硫酸肝素的生物合成中进行了关键的最后一步修饰。该酶将硫酰基（SO3）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，这是硫酸肝素与抗凝血酶结合的关键结构基序。在这项研究中，我们报道了3- o -硫代转移酶异构体1在2.5埃分辨率下与3'-磷酸腺苷5'-磷酸二元配合物的晶体结构。该结构揭示了3'-磷酸腺苷5'-硫酸磷酸结合的关键残基，并提示了硫酸肝素结合所需的残基。此外，位点定向诱变分析表明，残基Arg-67、Lys-68、Arg-72、Glu-90、His-92、Asp-95、Lys-123和Arg-276对酶活性至关重要。在这些必需氨基酸残基中，我们发现残基Arg-67、Arg-72、His-92和Asp-95在硫酸肝素3- o -硫基转移酶中保守，但在肝素n -去乙酰化酶/ n -硫基转移酶中不保守，这表明这些残基在赋予底物特异性方面发挥了作用。本研究结果为了解抗凝剂硫酸肝素的生物合成和硫酸肝素硫转移酶的一般作用机制提供了重要的信息。
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-Angstrom 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.