Inhibition of microbial β-N-acetylhexosaminidases by 4-deoxy- and galacto-analogues of NAG-thiazoline

NAG-thiazoline is a well-established competitive inhibitor of two physiologically relevant glycosidase families—β-N-acetylhexosaminidases (GH20) and β-N-acetylglucosaminidases (GH84). Based on the different substrate flexibilities of these enzyme groups, we designed and synthesized the 4-deoxy derivative of NAG-thiazoline aiming at the selective inhibition of GH20 β-N-acetylhexosaminidases. One GH84 and two GH20 microbial glycosidases were employed as model enzymes for the inhibition assays. Surprisingly, the new compound 4-deoxy-thiazoline exhibited no activity inhibition with either of the enzyme families of interest. Unlike with the substrates, the 4-hydroxyl group of the inhibitor’s sugar ring seems to be crucial for binding the inhibitor to the active sites of these enzymes.     

β-N-Acetylhexosaminidase: What's in a name…?

β-N-Acetylhexosaminidases (EC 3.2.1.52, belonging to CAZy GH families 3, 20 and 84) have recently gained a lot of attention, not only due to their implication in human physiology and disease, but also due to their great potential in the enzymatic synthesis of carbohydrates and glycomimetics. GH family 20 β-N-acetylhexosaminidases, and GH family 3 and 84 β-N-acetylglucosaminidases from all kinds of organisms have been intensively studied from the point of view of their physiological roles, reaction mechanisms, structure and inhibition. Thanks to their outstanding substrate promiscuity, extracellular β-N-acetylhexosaminidases from filamentous fungi are able to cleave and transfer substrates bearing various functionalities, ranging from carboxylates, sulfates, acylations to azides, and even 4-deoxy glycosides. Thus, they have proved to be versatile biosynthetic tools for the preparation of both natural and modified hexosaminides under mild conditions with good yields.     

Structure of N-acetyl-beta-D-glucosaminidase (GcnA) from the endocarditis pathogen Streptococcus gordonii and its complex with the mechanism-based inhibitor NAG-thiazoline

The crystal structure of GcnA, an N-acetyl-β-d-glucosaminidase from Streptococcus gordonii, was solved by multiple wavelength anomalous dispersion phasing using crystals of selenomethionine-substituted protein. GcnA is a homodimer with subunits each comprised of three domains. The structure of the C-terminal α-helical domain has not been observed previously and forms a large dimerisation interface. The fold of the N-terminal domain is observed in all structurally related glycosidases although its function is unknown. The central domain has a canonical (β/α)₈ TIM-barrel fold which harbours the active site. The primary sequence and structure of this central domain identifies the enzyme as a family 20 glycosidase. Key residues implicated in catalysis have different conformations in two different crystal forms, which probably represent active and inactive conformations of the enzyme. The catalytic mechanism for this class of glycoside hydrolase, where the substrate rather than the enzyme provides the cleavage-inducing nucleophile, has been confirmed by the structure of GcnA complexed with a putative reaction intermediate analogue, N-acetyl-β-d-glucosamine-thiazoline. The catalytic mechanism is discussed in light of these and other family 20 structures.