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 ␤-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. Heparin and heparan sulfate (HS)3 glycosaminoglycans (GAGs) are negatively charged, linear polysaccharides composed of repeating disaccharide units of uronic acid and glucosamine residues (GlcN, 2-amino-2-deoxy-␣-D-glucopyranose) (1). Heparin typically contains ϳ90% iduronic acid (IdoA, ␣-Lidopyranosyluronic acid) and 10% glucuronic acid (GlcA, ␤-Dglucopyranosyluronic acid), with a high content of 2-O-sulfo groups on the IdoA residue. The glucosamine residue in heparin is predominantly substituted with N-sulfo groups (GlcNS, where S is sulfo) and 6-O-sulfo groups with a small number of N-acetyl groups and much less frequently with 3-O-sulfo groups. In contrast, HS is somewhat more diverse in its primary structure and characterized by a higher percentage of the GlcA epimer, N-acetyl-substituted GlcN (GlcNAc) and a lower percentage of 2-O-sulfo, 6-O-sulfo, and N-sulfo groups. The modifications in HS are not uniform; rather, they are concentrated within specific regions of the polysaccharide, giving rise to a short, sulfo group containing sequence motifs responsible for the interactions between HS and a diverse repertoire of proteins leading to its multiple biological roles. These complex polysaccharides provide docking sites for numerous protein ligands involved in diverse biological processes, ranging from cancer and angiogenesis, anticoagulation, inflammatory processes, viral and microbial pathogenesis to multiple aspects of development (2-9). Moreover, HS-GAGs are abundant at the cell surface as part of the proteoglycan cell surface receptors (3, 4).Specialized microorganisms express GAG-degrading lyases serving nutritional purposes of both themselves and their vertebrat...

Structural basis of heparan sulfate-specific degradation by heparinase III

“…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 Polysaccharide Lyase from Stenotrophomonas maltophilia with a Unique, pH-regulated Substrate Specificity

“…The heparin activity of His 202 was attributed to the fact that the H5 proton of GlcA (found in heparin) points in the opposite direction of Tyr 257 , therefore preventing Tyr 257 from acting as a proton acceptor in GlcA containing substrates. Conversely the H5 proton of iduronic acid (found in heparan sulfate) points toward Tyr 257 , allowing the tyrosine to act as both proton acceptor and donor (62). In the case of Smlt1473, all three of the most active substrates contain GlcA (poly-GlcA and HA) or its C2 epimer ManA (poly-ManA), however, only HA contains GlcNAc.…”

“…In particular, Shaya et al (62) proposed a mechanism for heparinase II in which His 202 acted as proton acceptor in the cleavage of heparin and Tyr 257 acted as proton acceptor in the cleavage of heparan sulfate. Heparin contains mostly GlcNAc and GlcA, whereas heparin sulfate contains N-sulfoglucosamine (GlcNS) and iduronic acid, the C5 epimer of GlcA.…”

Insight into the Role of Substrate-binding Residues in Conferring Substrate Specificity for the Multifunctional Polysaccharide Lyase Smlt1473

“…A consensus has not been met regarding the specific roles of the catalytic histidine and tyrosine, namely whether tyrosine acts as proton acceptor and donor with histidine stabilizing an intermediate, the mechanism proposed for alginate lyases (4) and xanthan lyases (5), or whether histidine acts as the proton acceptor and tyrosine as the proton donor, the mechanism proposed for hyaluronan lyases (6,7). Furthermore, Shaya et al (8) proposed a third mechanism for heparinase II in which the residue responsible □ S This article contains supplemental Table S1. 1 To whom correspondence should be addressed: Dept.…”