Abstract

Unsaturated glucuronyl hydrolase (UGL), which is a member of glycoside hydrolase family GH-88, is a bacterial enzyme that degrades mammalian glycosaminoglycans and bacterial biofilms. The enzyme, which acts on unsaturated oligosaccharides with an alpha-glycoside bond produced by microbial polysaccharide lyases responsible for bacterial invasion of host cells, was believed to release 4-deoxy-l-threo-5-hexosulose-uronate (unsaturated glucuronic acid, or DeltaGlcA) and saccharide with a new nonreducing terminus by hydrolyzing the glycosidic bond. We detail the crystal structures of wild-type inactive mutant UGL of Bacillus sp. GL1 and its complex with a substrate (unsaturated chondroitin disaccharide), identify active site residues, and postulate a reaction mechanism catalyzed by UGL that triggers the hydration of the vinyl ether group in DeltaGlcA, based on the structural analysis of the enzyme-substrate complex and biochemical analysis. The proposed catalytic mechanism of UGL is a novel case among known glycosidases. Under the proposed mechanism, Asp-149 acts as a general acid and base catalyst to protonate the DeltaGlcA C4 atom and to deprotonate the water molecule. The deprotonated water molecule attacks the DeltaGlcA C5 atom to yield unstable hemiketal; this is followed by spontaneous conversion to an aldehyde (4-deoxy-l-threo-5-hexosulose-uronate) and saccharide through hemiacetal formation and cleavage of the glycosidic bond. UGL is the first clarified alpha(6)/alpha(6)-barrel enzyme using aspartic acid as the general acid/base catalyst.

Highlights

  • Glycosaminoglycans such as chondroitin, hyaluronan, and heparin are highly negatively charged polysaccharides with a repeating disaccharide unit consisting of a uronic acid residue and an amino sugar residue [1]

  • Unsaturated glucuronyl hydrolase (UGL) is the first clarified ␣6/␣6-barrel enzyme using aspartic acid as the general acid/ base catalyst

  • Chondroitin consists of D-glucuronic acid (GlcA) and N-acetyl-D-galactosamine (GalNAc) along with a sulfate group(s) at position 4 or 6 or at both positions

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Summary

Introduction

Glycosaminoglycans such as chondroitin, hyaluronan, and heparin are highly negatively charged polysaccharides with a repeating disaccharide unit consisting of a uronic acid residue (glucuronic or iduronic acid) and an amino sugar residue (glucosamine or galactosamine) [1]. GL1 and its complex with a substrate (unsaturated chondroitin disaccharide), identify active site residues, and postulate a reaction mechanism catalyzed by UGL that triggers the hydration of the vinyl ether group in ⌬GlcA, based on the structural analysis of the enzyme-substrate complex and biochemical analysis.

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