Potent inhibition of a GH20 exo-β-N-acetylglucosaminidase from marine Vibrio bacteria by reaction intermediate analogues

Abstract

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.

N‐acetyl‐β‐hexosaminidase activity is important for chitooligosaccharide metabolism and biofilm formation in Burkholderia pseudomallei

“…Bioinformatic analyses of B. pseudomallei bpsl0500 demonstrate conserved characteristics of GH20 N-acetyl-β-Dhexosaminidases B. pseudomallei bpsl0500 has been annotated as a member of the glycoside hydrolase (GH) family GH20 within the CAZy Database classification system (Lombard et al, 2014). Enzymes of this family utilise a substrate-assisted catalytic mechanism, for N-acetyl-β-D-hexosaminidases specifically this mechanism involves the participation of the 2-acetamido group of the substrate (Figure 1) (Mark et al, 2001;Meekrathok et al, 2018;Sumida et al, 2012;Tews, Perrakis, et al, 1996).…”

NAG‐thiazoline is a potent inhibitor of the Vibrio campbellii GH20 β‐N‐Acetylglucosaminidase

“…VhGlcNAcase is a chitin-recycling enzyme that helps to complete chitin degradation [17]. The catalytic mechanism of VhGlcNAcase involves substrate-assisted catalysis [42] and several compounds such as sodium azide [43] and the putative transition-state analogues PUGNAc, NHAcCAS, and NHAcDNJ have been previously shown to inhibit the activity of VhGlcNAcase [25], as well as that of other GH20 and GH84 GlcNAcases [28,33]. NGT bound to VhGlcNAcase with a K d of 32 AE 1.2 µM, as determined by ITC.…”

“…In an effort to gain insight into the biochemistry and biological function of GlcNAc-processing enzymes, small-molecule inhibitors have been developed and a number of inhibitors of both bacterial and human GH20 GlcNAcases have been reported. For example, PUGNAc (O-(2-acetamido-2-deoxy-Dglucopyranosylidene)amino N-phenylcarbamate) has been characterized as a potent inhibitor of GlcNAcases from not only family GH20 [25,26] but also GH3 [27] and GH84 [28]. NAG-thiazoline (1,2dideoxy-2ʹ-methyl-a-D-glucopyranoso-[2,1-d]-D2ʹ-thiazoline, NGT) [29] has been shown to be a potent inhibitor of both GH20 [30] and GH84 GlcNAcases [26] and iminosugars, such as 2-acetamido-1,2-dideoxynojirimycin (NHAcDNJ) and 6-acetamido-6-deoxy-castanospermine (NHAcCAS) have also been found to be the potent inhibitors of GlcNAcases from GH3 [31,32], GH20 [31,[33][34][35][36], and GH84 [28,31,36].…”

Purification and characterization of a chitinase from Aeromonas media CZW001 as a biocatalyst for producing chitinpentaose and chitinhexaose

“…Chitinases are widely produced in nature by some microorganisms, plants, and animals. Chitinases are classified into the glycosyl hydrolase (GH) families of GH18, GH19, and GH20, with different three‐dimensional structures and catalytic mechanisms 9,10 . There are also a few chitinases belonging to 23 families and 48 families 11 .…”

“…Chitinases are classified into the glycosyl hydrolase (GH) families of GH18, GH19, and GH20, with different three-dimensional structures and catalytic mechanisms. 9,10 There are also a few chitinases belonging to 23 families and 48 families. 11 The GH18 family is composed of chitinases from viruses, fungi, insects, plants, animals, etc.…”