Crystal Structure of Heparinase II from Pedobacter heparinus and Its Complex with a Disaccharide Product

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 Å resolution and in complex with a disaccharide product of heparin degradation at 2.30 Å resolution. The protein is composed of three domains: an N-terminal ␣-helical domain, a central twolayered ␤-sheet domain, and a C-terminal domain forming a twolayered ␤-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 Zn 2؉ 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.

Structural basis of heparan sulfate-specific degradation by heparinase III

“…Our study provides a representative structure of the PL12 family. Heparinase III shares a similar overall structure to heparinase II and Chondroitinase AC (ChonAC) which have been structurally well-characterized (Lunin et al, 2004;Shaya et al, 2006). These three proteins share a similar architecture composed of an (α/α) 5, 6 toroid and β-sandwich fold.…”

“…Two different approaches are employed to neutralize the negative charge on the carboxylic group. Pectin lyase utilizes a Ca 2+ ion to weaken the negative charge (Mayans et al, 1997) while others like HepII (Shaya et al, 2006) and ChonAC (Lunin et al, 2004) exert their asparagine residue to achieve the process. In our model, HepIII likely implements a similar strategy as ChonAC and HepII to accomplish the neutralization by employing its residue Asn260.…”

“…Both domains display distinguishable features compared to those of HepII ( Fig. 2B) (Shaya et al, 2006). In contrast, the N-terminal portion of HepI shrinks considerably, resulting in a β-jelly roll fold that is completely different from that of HepII and HepIII (Han et al, 2009).…”

“…Structure-based sequence alignment showed that the crucial residues in HepII related to degrading reaction are well-conserved in HepIII (Fig. 3) (Shaya et al, 2006(Shaya et al, , 2010. These important residues include Tyr314 and His464 in HepIII, and Tyr257 and His406 in HepII.…”

A Novel Structural Fold in Polysaccharide Lyases

“…These characteristics of lyases indicate that they share common structural features determining uronate recognition and reaction manner (␤-elimination). Crystal structures of polysaccharide lyases in 11 families have been determined thus far (Table 1), and structure and function relationships of enzymes such as lyases for pectate, pectin, alginate, chondroitin, hyaluronan, and xanthan are being analyzed (15,16,(27)(28)(29)(30)(31)(32). However, little knowledge has been accumulated on the mechanisms of substrate recognition and catalytic reaction of RG lyases.…”