title: Catalytic mechanism of heparinase II investigated by site-directed mutagenesis and the crystal structure with its substrate.
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.
authors.affiliation: Department of Biochemistry, McGill University, Montréal, Québec H3G 1Y6, Canada.
authors.forename: David
authors.initials: D
authors.lastname: Shaya
authors.forename: Wenjing
authors.initials: W
authors.lastname: Zhao
authors.forename: Marie-Line
authors.initials: ML
authors.lastname: Garron
authors.forename: Zhongping
authors.initials: Z
authors.lastname: Xiao
authors.forename: Qizhi
authors.initials: Q
authors.lastname: Cui
authors.forename: Zhenqing
authors.initials: Z
authors.lastname: Zhang
authors.forename: Traian
authors.initials: T
authors.lastname: Sulea
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Linhardt
authors.forename: Miroslaw
authors.initials: M
authors.lastname: Cygler
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis; D002240:Carbohydrate Sequence; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D004187:Disaccharides / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005417:Flavobacterium / Q000201:enzymology* / Q000235:genetics; D006493:Heparin / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004187:Disaccharides; D009844:Oligosaccharides; C464536:heparin disaccharide; D014443:Tyrosine; D006639:Histidine; D006493:Heparin; D011133:Polysaccharide-Lyases; C114663:heparinase II; D000409:Alanine
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