Dissecting conformational contributions to glycosidase catalysis and inhibition

Abstract

Glycoside hydrolases (GHs) are classified into >100 sequence-based families. These enzymes process a wide variety of complex carbohydrates with varying stereochemistry at the anomeric and other ring positions. The shapes that these sugars adopt upon binding to their cognate GHs, and the conformational changes that occur along the catalysis reaction coordinate is termed the conformational itinerary. Efforts to define the conformational itineraries of GHs have focussed upon the critical points of the reaction: substrate-bound (Michaelis), transition state, intermediate (if relevant) and product-bound. Recent approaches to defining conformational itineraries that marry X-ray crystallography of enzymes bound to ligands that mimic the critical points, along with advanced computational methods and kinetic isotope effects are discussed.

Graphical abstract

Figure 1

(a) Mechanisms of classical (i) inverting and retaining glycosidases that utilize (ii) an enzymic nucleophile or (iii) substrate-assisted catalysis. (b) Classical conformational itineraries around planar, oxocarbenium ion-like transition states in (i,ii) half-chair (H) or (iii,iv) boat (B) conformations. (c) Strategies and reagents used to study key species along the reaction coordinate.

Figure I

Figure 2

Computational studies, in concert with X-ray crystallography and inhibitor design and synthesis, assist in assigning conformational itineraries. (a) Assigning the conformational itinerary of Cellvibrio japonicas GH26 β-mannanase Man26C. (i) Free energy landscapes reveal mannoimidazole, unlike isofagomine, is able to attain the conformations relevant to glycosidase catalysis; (ii) X-ray structures of a Michaelis complex (1GVY), glycosyl enzyme intermediate (1GW1), and transition state mimicking β-mannosyl-1,4-mannoimidazole complex (4CD5); (iii) proposed conformational itinerary. (b) Assigning the conformational itinerary of Caulobacter strain K31 GH47 α-mannosidase. (i) Free energy landscapes highlight substrate preactivation off-enzyme, and reshaping of the available conformations on-enzyme; (ii) X-ray structures of Michaelis complex (4AYP), transition state mimicking mannoimidazole complex (4AYQ), and product mimicking noeuromycin complex (4AYR); (iii) proposed conformational itinerary.

Figure 3

New glycosidase conformational assignments. (a) A likely conformational itinerary for the GH39 human α-l-iduronidase based on structures of a Michaelis complex (4KGJ) and a glycosyl enzyme intermediate (4KH2). (b) A likely conformational itinerary for an α-l-neoagarobiase based on a Michaelis complex (4AK7). (c) A possible conformational itinerary for a GH99 endo-α-mannosidase based on a proposed mechanism that proceeds through a 1,2-anhydro sugar intermediate.

Figure 4

Conformational itinerary of influenza GH34 neuraminidases and conformational transition state mimicry by inhibitors. (a) The conformational itinerary of influenza GH34 neuraminidases informed by an X-ray structure of a glycosyl enzyme intermediate (4H52). (b) Complexes of anti-influenza drugs with influenza neuraminidases reveals that oseltamivir (2HU4), unlike zanamivir (1NNC), provides good conformational mimicry of the proposed sialidase transition state.