Bacterial chitobiase structure provides insight into catalytic mechanism and the basis of Tay–Sachs disease

Abstract

Chitin, the second most abundant polysaccharide on earth, is degraded by chitinases and chitobiases. The structure of Serratia marcescens chitobiase has been refined at 1.9 A resolution. The mature protein is folded into four domains and its active site is situated at the C-terminal end of the central (beta alpha)8-barrel. Based on the structure of the complex with the substrate disaccharide chitobiose, we propose an acid-base reaction mechanism, in which only one protein carboxylate acts as catalytic acid, while the nucleophile is the polar acetamido group of the sugar in a substrate-assisted reaction. The structural data lead to the hypothesis that the reaction proceeds with retention of anomeric configuration. The structure allows us to model the catalytic domain of the homologous hexosaminidases to give a structural rationale to pathogenic mutations that underlie Tay-Sachs and Sandhoff disease.

Purification, Cloning, and Sequence Analysis of β-N-Acetylglucosaminidase from the Chitinolytic BacteriumAeromonas hydrophilaStrain SUWA-9

“…Tews et al have reported that S. marcescens NAG uses an acid-base reaction mechanism with glutamic acid 540 as the catalytic amino acid. 12) This residue is well conserved in all members of family 20 of glycosyl hydrolases (Fig. 5).…”

Insight into a strategy for attenuating AmpC‐mediated β‐lactam resistance: Structural basis for selective inhibition of the glycoside hydrolase NagZ

“…Structural studies of N-acetyl-b-glucosaminidases from families GH3, 16 GH20, 20,21,24,25 and GH84 22,26 have revealed significant differences in the active site structures of enzymes carrying out substrate assisted catalysis versus those proceeding via a glycosyl-enzyme intermediate. For GH3 enzymes, it has been found that the 2-acetamido group of the substrate is not essential for cleavage of the glycosidic bond, consistent with these enzymes using an enzymic nucleophile.…”

“…Although GH3 enzymes, including NagZ, are functionally related to the GH20 human b-hexosaminidase isoenzymes and GH84 O-GlcNAcase, recent kinetic [17][18][19] and structural studies 16,[20][21][22] have revealed that GH3 enzymes use a catalytic mechanism that differs from that used by GH20 and GH84 enzymes. This distinction should therefore offer a tractable route to generating selective inhibitors of NagZ, and clear comparisons of these enzymes at a structural level would greatly accelerate these efforts.…”

“…1, path B). [19][20][21] In the first step of this mechanism, the carbonyl oxygen of the 2-acetamido group takes the place of an enzymic nucleophile, yielding a bicyclic oxazoline or oxazolinium ion intermediate. The nucleophilicity of the 2-acetamido group is enhanced by an essential catalytic residue that accepts a hydrogen-bond from this substituent.…”

Molecular cloning and sequence analysis of an ascidian egg β‐N‐acetylhexosaminidase with a potential role in fertilization

“…Alignment of the amino acid sequence of these ␤-hexosaminidases is shown in Figure 2. All of the residues, which are thought to form the active site of the subunits, suggested either from analyses of mutant human proteins (Brown & Mahuran 1991;Liessem et al 1995;Tse et al 1996;Fernandes et al 1997) or from structural analysis of a member of the glycosyl hydrolase family 20 (Serratia marcescens chitobiase; Tews et al 1996), are conserved in P. mammillata. We found five potential N-glycosylation sites in the P. mammillata ␤-hexosaminidase.…”