PDBsum entry: 3e7j

Sugar binding protein, lyase

PDB id: 3e7j

Contents

Protein chains

743 a.a. *

Ligands

ACT ×5

NAG-GCU-NAG-GCD ×2

Metals

_ZN ×2

Waters ×782

* Residue conservation analysis

PDB id:

3e7j

Name:

Sugar binding protein, lyase

Title:

Heparinaseii h202a/y257a double mutant complexed with a hepa sulfate tetrasaccharide substrate

Structure:

Heparinase ii protein. Chain: a, b. Fragment: unp residues 24-772. Mutation: yes

Source:

Pedobacter heparinus. Organism_taxid: 984

Resolution:

2.10Å R-factor: 0.201 R-free: 0.234

Authors:

D.Shaya,M.Cygler

Key ref:

D.Shaya et al. (2010). Catalytic mechanism of heparinase II investigated by site-directed mutagenesis and the crystal structure with its substrate. J Biol Chem, 285, 20051-20061. PubMed id: 20404324 DOI: 10.1074/jbc.M110.101071

Date:

18-Aug-08 Release date: 30-Dec-08

Protein chains

Q46080  (Q46080_PEDHE) -  Heparinase II protein (Precursor)

Seq: 772 a.a.

Struc: 743 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

DOI no: 10.1074/jbc.M110.101071

J Biol Chem 285:20051-20061 (2010)

PubMed id: 20404324

Catalytic mechanism of heparinase II investigated by site-directed mutagenesis and the crystal structure with its substrate.

D.Shaya, W.Zhao, M.L.Garron, Z.Xiao, Q.Cui, Z.Zhang, T.Sulea, R.J.Linhardt, M.Cygler.

ABSTRACT

Heparinase II (HepII) is an 85-kDa dimeric enzyme that depolymerizes both heparin and heparan sulfate glycosaminoglycans through a beta-elimination mechanism. Recently, we determined the crystal structure of HepII from Pedobacter heparinus (previously known as Flavobacterium heparinum) in complex with a heparin disaccharide product, and identified the location of its active site. Here we present the structure of HepII complexed with a heparan sulfate disaccharide product, proving that the same binding/active site is responsible for the degradation of both uronic acid epimers containing substrates. The key enzymatic step involves removal of a proton from the C5 carbon (a chiral center) of the uronic acid, posing a topological challenge to abstract the proton from either side of the ring in a single active site. We have identified three potential active site residues equidistant from C5 and located on both sides of the uronate product and determined their role in catalysis using a set of defined tetrasaccharide substrates. HepII H202A/Y257A mutant lost activity for both substrates and we determined its crystal structure complexed with a heparan sulfate-derived tetrasaccharide. Based on kinetic characterization of various mutants and the structure of the enzyme-substrate complex we propose residues participating in catalysis and their specific roles.