Substrate binding mode and catalytic mechanism of human heparan sulfate d -glucuronyl C5 epimerase

Abstract

Heparan sulfate (HS) is a linear, complex polysaccharide that modulates the biological activities of proteins through binding sites made by a series of Golgi-localized enzymes. Of these, glucuronyl C5-epimerase (Glce) catalyzes C5-epimerization of the HS component,d-glucuronic acid (GlcA), intol-iduronic acid (IdoA), which provides internal flexibility to the polymer and forges protein-binding sites to ensure polymer function. Here we report crystal structures of human Glce in the unbound state and of an inactive mutant, as assessed by real-time NMR spectroscopy, bound with a (GlcA-GlcNS)nsubstrate or a (IdoA-GlcNS)nproduct. Deep infiltration of the oligosaccharides into the active site cleft imposes a sharp kink within the central GlcNS-GlcA/IdoA-GlcNS trisaccharide motif. An extensive network of specific interactions illustrates the absolute requirement ofN-sulfate groups vicinal to the epimerization site for substrate binding. At the epimerization site, the GlcA/IdoA rings are highly constrained in two closely related boat conformations, highlighting ring-puckering signatures during catalysis. The structure-based mechanism involves the two invariant acid/base residues, Glu499 and Tyr578, poised on each side of the target uronic acid residue, thus allowing reversible abstraction and readdition of a proton at the C5 position through a neutral enol intermediate, reminiscent of mandelate racemase. These structures also shed light on a convergent mechanism of action between HS epimerases and lyases and provide molecular frameworks for the chemoenzymatic synthesis of heparin or HS analogs.

Through the Looking Glass: Chiral Recognition of Substrates and Products at the Active Sites of Racemases and Epimerases

“…[206] (L) The heparan components l-iduronic acid (l-IdoA,b lue) and d-glucuronic acid (d-GlcA,g reen)a sbound to GlcE from Homo sapiens (PDB 6I02 and 6I01, respectively)a re shown. [207] The red arrow denotesthe C5 stereocenter. coordinated to aM n 2 + ion.…”

“…[237][238][239][240] Recently,t he X-ray crystal structures of an inactivep ointmutant (Y578F) variant of soluble, recombinant humanG lcE (Val199-Asn617) with ab ound synthetic substrate ((GlcA-GlcNS) 5 ,P DB 6I01) and with ab ound synthetic product ((IdoA-GlcNS) 4 ,PDB 6I02) were reported. [207] The enzyme exists in solution as ad imer with each monomer composed of an N-terminal b-hairpin domain,ab barrel domain, and aC -terminal ahelical domain. The structures are consistentw ith an epimerization mechanism, which proceedsv ia abstraction of the aproton at C5 of GlcA/IdoA at the + 1s ubsite to form an acarbanion or enol(ate) intermediate that is subsequently reprotonated on the opposite face (Scheme 17).…”

“…[241][242][243] The phenolic oxygen of Tyr578 and the g-COO À of Glu499 have been proposed to acts as the Brønsted bases to abstract the C5 proton from the GlcA and IdoA units, respectively.D ebarnote tal. [207] proposed that the reaction intermediate is stabilized by general acid catalysis with transfer of ap roton from the side chain of Tyr560 to the oxygen of the carboxylate group of GlcA.…”

“…Both the negatively charged substrate and product are boundi nac urved, positively charged cleft that spans approximately 25-30 in length.T his cleft has as harp bend at the epimerization site, which is evident by the bent conformation of the polysaccharide chains in Figure 3L.T he sugar units of the substrate at subsites À1t o+ 3a re anchored firmly in place by multiple H-bonds and electrostatic interactions with the protein, particularly at the GlcNS moiety of the substrate in subsite À1, consistent with the absolute requirement of N-sulfate groups vicinal to the epimerizations ite for substrate binding. [207] Feweri nteractions are present between the non-reducing end of the ligandsa nd the protein at subsites À2t oÀ4. Overall,s uperposition of the structures of the bound epimers reveals that the corresponding sugar units of both the substrate and the product share the same positions ( Figure 3L).…”

“…These ring conformations differ from the less strained 4 C 1 chair conformationa nd 2 S O skew-boat conformation preferred by GlcA and IdoA, respectively. [207] Thus, the active site of GlcE has evolved to maintain am irror-image packing orientation of the bound enantiomers by allowing for movement of C5 andb ye nforcing the similar conformation of the sugar rings through multiple protein-ligandi nteractions at the + 1s ite and adjacent sites.…”