Qin Yi, Ke Jiyuan, Gu Xin, Fang Jianping, Wang Wucheng, Cong Qifei, Li Jie, Tan Jinzhi, Brunzelle Joseph S, Zhang Chenghai, Jiang Yi, Melcher Karsten, Li Jin-Ping, Xu H Eric, Ding Kan
From the Glycochemistry and Glycobiology Laboratory, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Pudong, Shanghai 201203, China,; the VARI-SIMM Center, Center for Structure and Function of Drug Targets, Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Laboratory of Structural Sciences, Center for Structural Biology and Drug Discovery, Van Andel Research Institute, Grand Rapids, Michigan 49503,.
J Biol Chem. 2015 Feb 20;290(8):4620-4630. doi: 10.1074/jbc.M114.602201. Epub 2015 Jan 7.
Heparan sulfate (HS) is a glycosaminoglycan present on the cell surface and in the extracellular matrix, which interacts with diverse signal molecules and is essential for many physiological processes including embryonic development, cell growth, inflammation, and blood coagulation. D-glucuronyl C5-epimerase (Glce) is a crucial enzyme in HS synthesis, converting D-glucuronic acid to L-iduronic acid to increase HS flexibility. This modification of HS is important for protein ligand recognition. We have determined the crystal structures of Glce in apo-form (unliganded) and in complex with heparin hexasaccharide (product of Glce following O-sulfation), both in a stable dimer conformation. A Glce dimer contains two catalytic sites, each at a positively charged cleft in C-terminal α-helical domains binding one negatively charged hexasaccharide. Based on the structural and mutagenesis studies, three tyrosine residues, Tyr(468), Tyr(528), and Tyr(546), in the active site were found to be crucial for the enzymatic activity. The complex structure also reveals the mechanism of product inhibition (i.e. 2-O- and 6-O-sulfation of HS keeps the C5 carbon of L-iduronic acid away from the active-site tyrosine residues). Our structural and functional data advance understanding of the key modification in HS biosynthesis.
硫酸乙酰肝素（HS）是一种存在于细胞表面和细胞外基质中的糖胺聚糖，它与多种信号分子相互作用，对包括胚胎发育、细胞生长、炎症和血液凝固在内的许多生理过程至关重要。D-葡萄糖醛酸C5-表异构酶（Glce）是HS合成中的一种关键酶，它将D-葡萄糖醛酸转化为L-艾杜糖醛酸以增加HS的灵活性。HS的这种修饰对于蛋白质配体识别很重要。我们已经确定了apo形式（未结合配体）的Glce以及与肝素六糖（Glce经O-硫酸化后的产物）形成复合物的晶体结构，二者均处于稳定的二聚体构象。一个Glce二聚体包含两个催化位点，每个位点位于C端α-螺旋结构域中一个带正电荷的裂隙处，结合一个带负电荷的六糖。基于结构和诱变研究，发现活性位点中的三个酪氨酸残基Tyr(468)、Tyr(528)和Tyr(546)对酶活性至关重要。复合物结构还揭示了产物抑制的机制（即HS的2-O-和6-O-硫酸化使L-艾杜糖醛酸的C5碳远离活性位点的酪氨酸残基）。我们的结构和功能数据推动了对HS生物合成中关键修饰的理解。
