3E7J

HeparinaseII H202A/Y257A double mutant complexed with a heparan sulfate tetrasaccharide substrate

Summary for 3E7J

• Entry DOI: 10.2210/pdb3e7j/pdb
• Related: 3E80
• Descriptor: Heparinase II protein, 4-deoxy-alpha-L-threo-hex-4-enopyranuronic acid-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-alpha-D-glucopyranuronic acid-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose, ZINC ION, ... (5 entities in total)
• Functional Keywords: alpha and beta lyase, alpha6/alpha6 incomplete toroid, sugar binding protein, lyase
• Biological source: Pedobacter heparinus
• Total number of polymer chains: 2
• Total formula weight: 171576.37

Authors

Shaya, D.,Cygler, M. (deposition date: 2008-08-18, release date: 2008-12-30, Last modification date: 2023-08-30)

Primary citation

Shaya, D.,Zhao, W.,Garron, M.L.,Xiao, Z.,Cui, Q.,Zhang, Z.,Sulea, T.,Linhardt, R.J.,Cygler, M.
Catalytic mechanism of heparinase II investigated by site-directed mutagenesis and the crystal structure with its substrate.
J.Biol.Chem., 285:20051-20061, 2010

PubMed 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.

PubMed: 20404324
DOI: 10.1074/jbc.M110.101071

Experimental method

X-RAY DIFFRACTION (2.1 Å)