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

Loading...
Thumbnail Image
Authors
Shaya, D.
Zhao, W.
Garron, M.L.
Xiao, Z.
Cui, Q.
Zhang, Z.
Sulea, T.
Linhardt, Robert J.
Cygler, M.
Issue Date
2010
Type
Article
Language
en_US
Keywords
Biology , Chemistry and chemical biology , Chemical and biological engineering , Biomedical engineering
Research Projects
Organizational Units
Journal Issue
Alternative Title
Abstract
Heparinase II (HepII) is an 85-kDa dimeric enzyme that depolymerizes both heparin and heparan sulfate glycosaminoglycans through a β-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.
Description
Journal of Biological Chemistry, 285, 20051-20061
Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.
Full Citation
Catalytic mechanism of heparinase II revealed 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, Journal of Biological Chemistry, 285, 20051-20061, 2010.
Publisher
Elsevier
Journal
Volume
Issue
PubMed ID
DOI
ISSN
EISSN