BackgroundVibrio harveyi GH20 β-N-acetylglucosaminidase (VhGlcNAcase) is a chitinolytic enzyme responsible for the successive degradation of chitin fragments to GlcNAc monomers, activating the onset of the chitin catabolic cascade in marine Vibrios.MethodsTwo invariant acidic pairs (Asp303-Asp304 and Asp437-Glu438) of VhGlcNAcase were mutated using a site-directed mutagenesis strategy. The effects of these mutations were examined and the catalytic roles of these active-site residues were elucidated using a chemical rescue approach. Enhancement of the enzymic activity of the VhGlcNAcase mutants was evaluated by a colorimetric assay using pNP-GlcNAc as substrate.ResultsSubstitution of Asp303, Asp304, Asp437 or Glu438 with Ala/Asn/Gln produced a dramatic loss of the GlcNAcase activity. However, the activity of the inactive D437A mutant was recovered in the presence of sodium formate. Our kinetic data suggest that formate ion plays a nucleophilic role by mimicking the β-COO-side chain of Asp437, thereby stabilizing the reaction intermediate during both the glycosylation and the deglycosylation steps.ConclusionsChemical rescue of the inactive D437A mutant of VhGlcNAcase by an added nucleophile helped to identify Asp437 as the catalytic nucleophile/base, and hence its acidic partner Glu438 as the catalytic proton donor/acceptor.General SignificanceIdentification of the catalytic nucleophile of VhGlcNAcases supports the proposal of a substrate-assisted mechanism of GH20 GlcNAcases, requiring the catalytic pair Asp437-Glu438 for catalysis. The results suggest the mechanistic basis of the participation of β-N-acetylglucosaminidase in the chitin catabolic pathway of marine Vibrios.
Vibrio harveyi -N-acetylglucosaminidase (VhGlcNAcase) is a new member of the GH20 glycoside hydrolase family responsible for the complete degradation of chitin fragments, with N-acetylglucosamine (GlcNAc) monomers as the final products. In this study, the crystallization and preliminary crystallographic data of wild-type VhGlcNAcase and its catalytically inactive mutant D437A in the absence and the presence of substrate are reported. Crystals of wild-type VhGlcNAcase were grown in 0.1 M sodium acetate pH 4.6, 1.4 M sodium malonate, while crystals of the D437A mutant were obtained in 0.1 M bis-tris pH 7.5, 0.1 M sodium acetate, 20% PEG 3350. X-ray data from the wild-type and the mutant crystals were collected at a synchrotron-radiation light source and were complete to a resolution of 2.5 Å . All crystals were composed of the same type of dimer, with the substrate N,N 0 -diacetylglucosamine (GlcNAc 2 or diNAG) used for soaking was cleaved by the active enzyme, leaving only a single GlcNAc molecule bound to the protein.
Exo-β-N-acetylglucosaminidases (GlcNAcases) are hydrolytic enzymes involved in the metabolism of chitin in bacteria and in eukaryotic glycosphingolipid metabolism, with genetic defects in human GlcNAcases (HexA and HexB) resulting in Tay-Sachs and Sandhoff diseases, respectively. Here, we determined the effects of three known inhibitors of exo-β-N-acetylglucosaminidases (PUGNAc, NHAcCAS and NHAcDNJ) on a GH20 exo-β-N-GlcNAcase (VhGlcNAcase) from the pathogenic bacterium Vibrio harveyi, in dose-response experiments. The inhibitors were shown to modify the kinetic parameters (both K and k), yielding significant decreases in the overall efficiency of the enzyme in hydrolyzing the natural substrate diNAG. Molecular interactions between the inhibitors and the enzyme were investigated by isothermal calorimetry (ITC), and were confirmed using molecular docking. VhGlcNAcase was strongly inhibited by these compounds, with PUGNAc having the lowest IC value, of 1.2 μM. Molecular docking suggested that the inhibitors mimicked reaction intermediates, with enzyme-inhibitor interactions being similar to those of the enzyme with diNAG. The equilibrium dissociation constants (K) obtained from ITC were 0.19 μM for PUGNAc, 12.9 μM for NHAcCAS and 25.6 μM for NHAcDNJ, confirming that PUGNAc was the most potent inhibitor. The ITC data indicated that the binding of the enzyme to the inhibitors was driven by enthalpy. The negative heat capacity change (ΔC) of -0.34 ± 0.05 kcal·mol·K indicates that hydrophobic interactions make a substantial contribution to the molecular interactions between PUGNAc and the enzyme. Our results suggest that PUGNAc is a highly potent inhibitor, and suggest its usefulness as a scaffold for potential drugs targeting GlcNAcase-related metabolic diseases.
GH-18 chitinases are chitinolytic enzymes, primarily responsible for the recycling of insoluble chitin biomaterials. These enzymes contain three invariant acidic active-site residues within a DXDXE motif, which play a synergistic role in the catalytic cycle of chitin degradation. We employed a pK calculation approach to approximate the protonation states of residues D1, D2, and E in the DXDXE motif of 75 GH-18 chitinases. Theoretical pH-activity profiles of these enzymes were subsequently constructed and compared with the experimentally determined pH-activity profiles. Theoretical pK data indicate that in the majority of chitinases the D1 side-chain is in the "up" and the E side-chain in the "down" position, while the position of the D2 side-chain is versatile and depends on the state of the enzyme. The pK values in 75 GH-18 chitinases were predicted to be <0 for D1, 8-13 for D2, and 6-9 for E, indicating that the D1-D2 pair holds exactly one net negative charge. On the other hand, the catalytic acid E is protonated over the active pH-range, agreeing with the pH-activity curves reported previously for most chitinases. The results obtained from this study help to elucidate the mechanistic details of the concerted participation of D1, D2, and E in the catalytic cycle of chitin hydrolysis by GH-18 chitinases.
Vibrio spp. play a vital role in the recycling of chitin in oceans, but several Vibrio strains are highly infectious to aquatic animals and humans. These bacteria require chitin for growth; thus, potent inhibitors of chitin-degrading enzymes could serve as candidate drugs against Vibrio infections. This study examined NAG-thiazoline (NGT)-mediated inhibition of a recombinantly expressed GH20 b-N-acetylglucosaminidase, namely VhGlcNAcase from Vibrio campbellii (formerly V. harveyi) ATCC BAA-1116. NGT strongly inhibited VhGlcNAcase with an IC 50 of 11.9 AE 1.0 lM and K i 62 AE 3 µM, respectively. NGT was also found to completely inhibit the growth of V. campbellii strain 650 with an minimal inhibitory concentration value of 0.5 µM. ITC data analysis showed direct binding of NGT to VhGlcNAcase with a K d of 32 AE 1.2 lM. The observed DG°b inding of À7.56 kcalÁmol À1 is the result of a large negative enthalpy change and a small positive entropic compensation, suggesting that NGT binding is enthalpy-driven. The structural complex shows that NGT fully occupies the substrate-binding pocket of VhGlcNAcase and makes an exclusive hydrogen bond network, as well as hydrophobic interactions with the conserved residues around the À1 subsite. Our results strongly suggest that NGT could serve as an excellent scaffold for further development of antimicrobial agents against Vibrio infections.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.