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

European Molecular Biology Laboratory, Hamburg, Germany.
Nature Structural Biology 08/1996; 3(7):638-48. DOI: 10.1038/nsb0796-638
Source: PubMed


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.

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    • "catalyze the removal of terminal N-acetylglucosamine (GlcNAc) or N-acetylgalactosamine (GalNAc) residues from various GlcNAc/GalNAc-containing glycans or glycolipids. Crystallographic information has shown that these enzymes employ a substrate-assisted mechanism1234567891011. To handle physiological substrates with different glycosidic linkages (β1,2, β1,3, β1,4 or β1,6) or different architectures (linear or branched, free or conjugated with lipids and proteins), GH20 β-GlcNAcases have evolved to show different substrate preferences5111213. "
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    ABSTRACT: Selective inhibition of function-specific β-GlcNAcase has great potential in terms of drug design and biological research. The symmetrical bis-naphthalimide M-31850 was previously obtained by screening for specificity against human glycoconjugate-lytic β-GlcNAcase. Using protein-ligand co-crystallization and molecular docking, we designed an unsymmetrical dyad of naphthalimide and thiadiazole, Q2, that changes naphthalimide specificity from against a human glycoconjugate-lytic β-GlcNAcase to against insect and bacterial chitinolytic β-GlcNAcases. The crystallographic and in silico studies reveal that the naphthalimide ring can be utilized to bind different parts of these enzyme homologs, providing a new starting point to design specific inhibitors. Moreover, Q2-induced closure of the substrate binding pocket is the structural basis for its 13-fold increment in inhibitory potency. Q2 is the first non-carbohydrate inhibitor against chitinolytic β-GlcNAcases. This study provides a useful example of structure-based rationally designed inhibitors as potential pharmaceuticals or pesticides.
    Scientific Reports 08/2014; 4:6188. DOI:10.1038/srep06188 · 5.58 Impact Factor
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    • "The structure model of the catalytic domain of OfHex3 was built using the catalytic domain of OfHex1 (PDB ID: 3NSN) as a template and was validated using PROCHECK (89.8% of the residues were in the most favored regions) and Verify_3D (93.4% of the residues had an average three- to one-dimensional score >0.2). The coordinates of (GlcNAc)2 were built based on the structure of the (GlcNAc)2 complexed chitobiase from Serratia marcescens (PDB ID: 1QBB) [33]. The structures of the active pockets of OfHex1 and OfHex3 were then compared (Fig. 1C). "
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    ABSTRACT: Insects require molting fluids to shed the old cuticle during molting. β-N-acetyl-D-hexosaminidase, known as Hex1, together with various chitinases, is responsible for degrading the chitin component of the old cuticle. This study showed that another β-N-acetyl-D-hexosaminidase, termed OfHex3, interacted with Hex1 and functioned in the molting fluid, although the homolog of OfHex3 was known as a sperm-plasma enzyme functioning in egg-sperm recognition. OfHex3 is an enzyme cloned from the insect Asian corn borer, Ostrinia furnacalis, which is one of the most destructive pests of maize. The enzymatic activity analysis indicated that OfHex3 was able to degrade chitooligosaccharides, but at a lower rate than that of OfHex1. Because OfHex3 did not have substrate inhibition, we deduced that the presence of OfHex3 might help OfHex1 relieve substrate inhibition during chitin degradation during molting. The expression patterns of OfHex3 during O. furnacalis development were studied by real-time PCR as well as western blot. The results showed that both gene transcription and protein translation levels of OfHex3 were up-regulated during larval-larval molting. The tissue-specific expression pattern analysis indicated that OfHex3 was mostly localized in the fat body and testis. All these data further supported that Hex3 was involved in molting as well as in fertilization. This study may help to understand the complexity of cuticle degradation during insect molting, and may provide a possible target for pest control.
    PLoS ONE 08/2013; 8(8):e71738. DOI:10.1371/journal.pone.0071738 · 3.23 Impact Factor
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    • "These five proteins were well characterized and shown to display chitinolytic activity. Recently, the crystal structures of these enzymes were reported on chitinase (ChiA and ChiB) and chitobiase (Perrakis et al., 1994; Tews et al., 1996; Van Aalten et al., 2000). The potential use of antibodies to assess chitinase produced from S. marcescens has not been extensively studied. "
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    ABSTRACT: In this study, a bacterium Serratia marcescens PRC-5 that displayed strong chitinolytic activity on 0.5% colloidal chitin-containing agar medium was isolated from soil. The chitinase activity increased rapidly with a maximum level (6.14U/mL) on 4 days of incubation with swollen chitin (pH 5.0). Three active bands of chitinase isozymes were observed (53, 44, and 34kDa) on SDS-PAGE gel and there pI values ranged from pI 5.4 to 5.8 on 2D gels. The chitinase from the PRC-5 strain was also able to produce GlcNAc monomers on TLC plates. The chitinase of PRC-5 inhibited the mycelial growth of Rhizoctonia solani KACC40111, which indicates that it could be used as a biocontrol agent for phytophathogens. The chitinase isozyme N1, which had a molecular weight of 62kDa, was transferred from a native and SDS-PAGE gel onto an immunoblot and was probed using an anti-PrGV-chitinase.
    02/2013; 92(2):2276-81. DOI:10.1016/j.carbpol.2012.12.018
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