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747 a.a.
_ZN
Key reference
DOI no: 10.1074/jbc.M512055200 J Biol Chem 281:15525-15535 (2006) PubMed id: 16565082
Crystal structure of heparinase II from Pedobacter heparinus and its complex with a disaccharide product. D.Shaya, A.Tocilj, Y.Li, J.Myette, G.Venkataraman, R.Sasisekharan, M.Cygler. ABSTRACT
Heparinase II depolymerizes heparin and heparan sulfate glycosaminoglycans, yielding unsaturated oligosaccharide products through an elimination degradation mechanism. This enzyme cleaves the oligosaccharide chain on the nonreducing end of either glucuronic or iduronic acid, sharing this characteristic with a chondroitin ABC lyase. We have determined the first structure of a heparin-degrading lyase, that of heparinase II from Pedobacter heparinus (formerly Flavobacterium heparinum), in a ligand-free state at 2.15 A resolution and in complex with a disaccharide product of heparin degradation at 2.30 A resolution. The protein is composed of three domains: an N-terminal alpha-helical domain, a central two-layered beta-sheet domain, and a C-terminal domain forming a two-layered beta-sheet. Heparinase II shows overall structural similarities to the polysaccharide lyase family 8 (PL8) enzymes chondroitin AC lyase and hyaluronate lyase. In contrast to PL8 enzymes, however, heparinase II forms stable dimers, with the two active sites formed independently within each monomer. The structure of the N-terminal domain of heparinase II is also similar to that of alginate lyases from the PL5 family. A Zn2+ ion is bound within the central domain and plays an essential structural role in the stabilization of a loop forming one wall of the substrate-binding site. The disaccharide binds in a long, deep canyon formed at the top of the N-terminal domain and by loops extending from the central domain. Based on structural comparison with the lyases from the PL5 and PL8 families having bound substrates or products, the disaccharide found in heparinase II occupies the "+1" and "+2" subsites. The structure of the enzyme-product complex, combined with data from previously characterized mutations, allows us to propose a putative chemical mechanism of heparin and heparan-sulfate degradation.
Selected figure(s)
Figure 1.
FIGURE 1. Stereo view of the schematic representation of heparinase II. A, monomer, with N-terminal domain (salmon), central domain (blue), and C-terminal domain (orange). The product is shown in a CPK representation, and the Zn^2+ ion is shown as a yellow sphere; B, N-terminal domain colored blue to red from the N to C terminus. Secondary structure elements are marked. C, middle domain, colored as in b; D, C-terminal domain, colored as in b; E, dimer, the first molecule colored as in a and the second molecule colored in shades of green. On the left is the schematic representation, and on the right is the surface representation. The C-terminal domain of one molecule packs into a depression between the central and the C-terminal domains of the other molecule. This figure was prepared using PyMol (available on the World Wide Web at www.pymol.org).Figure 3.
FIGURE 3. Substrate binding site. A, disaccharide productUAp2S(1–4)GlcNS6S with electron density ("omit map" calculated with phases derived from the model without the disaccharide) contoured at the 3
level. B, surface representation of the binding site with a disaccharide product shown in a ball-and-stick representation. The N-terminal domain is shown in magenta, and the central domain is orange. The product occupies the plus sites, and the minus sites are empty. C, ball-and-stick representation of the disaccharide product bound to HepII. The disaccharide is shown in thicker lines, its carbon atoms are green, the surrounding HepII residues are shown in thin lines, and their carbon atoms are gray. The hydrogen bonds are shown by dashed lines and are colored yellow when between the disaccharide and protein residues and cyan between the protein residues. The red spheres represent water molecules participating in the hydrogen bonding network. D, schematic representation of interactions between the disaccharide product and HepII. The residues shown in gray approach the sugars from below. Three parallel lines indicate stacking of a side chain with the sugar ring. Substituents in positions 1, 2, and 3 of the uronic acid are in axial orientations in our structure.
The above figures are reprinted by permission from the ASBMB: J Biol Chem (2006, 281, 15525-15535) copyright 2006. Figures were selected by an automated process.
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