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Probing the detailed interaction between lytic polysaccharide monooxygenases (LPMOs) and their polysaccharide substrates is key to revealing further insights into the mechanism of action of this class of enzymes on recalcitrant biomass. This investigation is somewhat hindered, however, by the insoluble nature of the substrates, which precludes the use of most optical spectroscopic techniques. Herein, we report a new semi-oriented EPR method which evaluates directly the binding of cellulose-active LPMOs to crystalline cellulose. We make use of the intrinsic order of cellulose fibres in Apium graveolens (celery) to orient the LPMO with respect to the magnetic field of an EPR spectrometer. The subsequent angle-dependent changes observed in the EPR spectra can then be related to the orientation of the g matrix principal directions with respect to the magnetic field of the spectrometer and, hence, to the binding of the enzyme onto the cellulose fibres. This method, which does not require specific modification of standard CW-EPR equipment, can be used as a general procedure to investigate LPMO–cellulose interactions.

A set of mannuronic acid based iminosugars, comprising the C-5-carboxylic acid, -methyl ester and -amide analogues of 1-deoxymannorjirimicin (DMJ), was synthesized and their pH dependent conformational behavior studied. Under acidic conditions the methyl ester and the carboxylic acid took up an "inverted" 1C4 chair conformation as opposed to the "normal" 4C1 chair at basic pH. This conformational change is explained by the stereoelectronic effects of the ring substituents and it parallels the behavior of the mannuronic acid ester oxocarbenium ion. Because of this solution phase behavior, the mannuronic acid ester azasugar was probed as an inhibitor for a Caulobacter GH47 mannosidase that hydrolyzes its substrates following a reaction itinerary that proceeds through a 3H4 transition state. No binding was observed for the mannuronic acid ester azasugar, but sub-atomic resolution data were obtained for the DMJ-CkGH47 complex, showing two conformations, 3S1 and 1C4, for the DMJ inhibitor.

Conformational analysis of enzyme-catalyzed mannoside hydrolysis has revealed two predominant conformational itineraries through B 2,5 or 3H 4 transitionstate (TS) conformations. A prominent unassigned catalytic itinerary is that of exo-1,6-α-mannosidases belonging to CAZy family 125. A published complex of Clostridium perfringens GH125 enzyme with a nonhydrolyzable 1,6-α-thiomannoside substrate mimic bound across the active site revealed an undistorted 4 C 1 conformation and provided no insight into the catalytic pathway of this enzyme. We show through a purely computational approach (QM/MM metadynamics) that sulfur-for-oxygen substitution in the glycosidic linkage fundamentally alters the energetically accessible conformational space of a thiomannoside when bound within the GH12S active site. Modeling of the conformational free energy landscape (FEL) of a thioglycoside strongly favors a mechanistically uninformative 4 C 1 conformation within the GH125 enzyme active site, but the FEL of corresponding O-glycoside substrate reveals a preference for a Michaelis complex in an oS 2 conformation (consistent with catalysis through a B 2,5 TS). This prediction was tested experimentally by determination of the 3D X-ray structure of the pseudo-Michaelis complex of an inactive (D220N) variant of C. perfringens GH125 enzyme in complex with 1,6-α-mannobiose. This complex revealed unambiguous distortion of the -1 subsite mannoside to an oS 2 conformation, matching that predicted by theory and supporting an oS 2 → B 2,5 → 1S 5 conformational itinerary for GH125 α-mannosidases. This work highlights the power of the QM/MM approach and identified shortcomings in the use of nonhydrolyzable substrate analogues for conformational analysis of enzymebound species.

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.