Structural and Functional Study of d-Glucuronyl C5-epimerase

Joseph S Brunzelle,Karsten Melcher,Jianping Fang,Wucheng Wang,Jinzhi Tan,Jiyuan Ke,Yi Qin,Qifei Cong,Jin-Ping Li,Jie Li,H.Eric Xu,Xin Gu,Chenghai Zhang,Yi Jiang,Kan Ding

doi:10.1074/jbc.m114.602201

Abstract

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.

Highlights

D-Glucuronyl C5-epimerase is a crucial modifying enzyme in the heparan sulfate biosynthesis pathway
Because heparin hexasaccharide can still bind to the active site of Glucuronyl C5-epimerase (Glce) (Fig. 3G), we examined whether the product after consecutive modification by Glce and O-sulfotransferases might influence the activity of Glce using a soluble in vitro reaction system
A long Heparan sulfate (HS) chain may thread through the two active sites, or two such polysaccharide chains may simultaneously bind to two active sites of the Glce dimer to allow HS modifications

Summary

Background

D-Glucuronyl C5-epimerase is a crucial modifying enzyme in the heparan sulfate biosynthesis pathway. 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. D-Glucuronyl C5-epimerase (Glce) is a key enzyme in HS/heparin synthesis, converting D-glucuronic acid (GlcA) to L-iduronic acid (IdoA) by C5 epimerization at the polymer level [4] (Fig. 1B). We report the first crystal structures of Glce, in apo-form (unliganded) and in complex with heparin hexasaccharide, which is the product of Glce following 2-O-sulfation on iduronic acid and 6-O-sulfation on N-sulfoglucosamine.

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION