Structural and biochemical evidence for a boat-like transition state in β-mannosidases

Abstract

Enzyme inhibition through mimicry of the transition state is a major area for the design of new therapeutic agents. Emerging evidence suggests that many retaining glycosidases that are active on alpha- or beta-mannosides harness unusual B2,5 (boat) transition states. Here we present the analysis of 25 putative beta-mannosidase inhibitors, whose Ki values range from nanomolar to millimolar, on the Bacteroides thetaiotaomicron beta-mannosidase BtMan2A. B2,5 or closely related conformations were observed for all tightly binding compounds. Subsequent linear free energy relationships that correlate log Ki with log Km/kcat for a series of active center variants highlight aryl-substituted mannoimidazoles as powerful transition state mimics in which the binding energy of the aryl group enhances both binding and the degree of transition state mimicry. Support for a B2,5 transition state during enzymatic beta-mannosidase hydrolysis should also facilitate the design and exploitation of transition state mimics for the inhibition of retaining alpha-mannosidases--an area that is emerging for anticancer therapeutics.

Catalytic Mechanism of Human α-Galactosidase

“…Thus, a new class of inhibitors for ␣-GAL (and other related family 27 glycosidases) might adopt a 4 H 3 half-chair (or the closely related 4 E envelope) conformation of the ring. Transition state mimics have not been developed for ␣-GAL, however, transition state analogues have been developed as selective inhibitors of mannosidases and other glycosidases (45,47,48).…”

Insights into the structure and function of fungal β‐mannosidases from glycoside hydrolase family 2 based on multiple crystal structures of the Trichoderma harzianum enzyme

“…To date, BtMan2A is the only structurally characterized bacterial GH family 2 b-mannosidase [19][20][21]. The enzyme has a complex five-domain fold with a central catalytic a/b-barrel domain surrounded by four smaller b-domains, which seems to be the typical tertiary structure architecture for GH family 2 enzymes.…”

Mechanism of Cellulose Hydrolysis by Inverting GH8 Endoglucanases: A QM/MM Metadynamics Study

“…5,6 The conformational interconversions that occur in the active sites of specific GHs have been the subject of many recent studies. [7][8][9][10][11][12][13][14] Nevertheless, direct evidence of conformational itineraries is still missing for many GH families. Furthermore, detailed structural features of the enzymatic transition state (TS) in GHs are still fairly unknown.…”

“…Most of the information that we have about TS structures in GHs comes from kinetic isotope effect measurements and structural information of GHs complexed with TS mimics. 12,15,16 Fortunately, reliable computational techniques can be exploited to explicitly model reactions inside GHs. To date, the reaction mechanism in GHs has been modeled only for GH22 lysozyme and GH2 -galactosidase by first-principles approaches, providing evidence of a covalent glycosyl-enzyme intermediate for these retaining GHs.…”