Catalytic mechanism of heparinase II revealed by site-directed mutagenesis and the crystal structure with its substrate
AuthorShaya, D.; Zhao, W.; Garron, M.L.; Xiao, Z.; Cui, Q.; Zhang, Z.; Sulea, T.; Linhardt, Robert J.; Cygler, M.
SubjectBiology; Chemistry and chemical biology; Chemical and biological engineering; Biomedical engineering
Full CitationCatalytic 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.
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AbstractHeparinase 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.;
DescriptionJournal 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.
DepartmentThe Linhardt Research Labs.; The Shirley Ann Jackson, Ph.D. Center for Biotechnology and Interdisciplinary Studies (CBIS);
RelationshipsThe Linhardt Research Labs Online Collection; Rensselaer Polytechnic Institute, Troy, NY; https://harc.rpi.edu/;
AccessOpen Access; A full text version is available in DSpace@RPI; A full text version is available in DSpace@RPI;
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