Substrate Distortion by aβ-Mannanase: Snapshots of the Michaelis and Covalent-Intermediate Complexes Suggest aB2,5 Conformation for the Transition State

Abstract

More than 6000 glycosidase sequences and related open reading frames are currently known. They have been classified into some 85 families based upon amino acid sequence similarities [1] providing a rich context in which to explore variations in glycosidase mechanism. Experimental demonstration of pyranoside ring conformations along the reaction pathway may assist the design of transition state analogues both as therapeutic agents and mechanistic probes. Here we report the three-dimensional structures of the Michaelis complex and covalent glycosyl ± enzyme intermediate for a family-26 b-mannanase, both of which display conformational features never previously seen on any glycosidase. When viewed in light of published work on mannosidase inhibition, this work suggests that the transition state for mannoside conditions to those described in Figure 4. These magnetic data are very similar to recent experimental results reported by other groups on complexes of Cu 2 dimers. [26] Theoretical fit using the BB model to the temperature-dependent molar susceptibility data in this case yields fit parameters J À 380 cm À1 and J' À 85 cm À1 . Clearly, the most striking feature is that the field-dependent magnetization does not display a hysteresis loop in this system, the straight line obtained being representative of a more traditional paramagnetic behavior.Our results dramatically underscore the potential afforded by supramolecular chemistry for the design of molecular nanostructured assemblies with desirable physical properties, while emphasizing how the composition of a material is not the only feature one must consider when designing a phase that exhibits molecular magnetism. Future work will focus on the modularity of this system and on chemical modification of the components: substituting the metal; changing the coordinated ligand; substituting the bdc ligand; incorporation of different guest molecules. We expect a significant effect on magnetic properties as it has already been shown that simply varying the apical coordinated ligand has a measurable effect on the magnetism exhibited by the SBU used in our study. Intensity data for 1 were collected at 173 K on a Bruker SMART-APEX diffractometer using Mo Ka radiation (l 0.7107 ä): Cu 6 C 123.08 H 54 N 9.37 O 30.73 , 2536.69 g mol À1 , trigonal, space group P3 ≈ c1, a b 18.6523(11), c 19.8313(18) ä, V 5975.1(7) ä 3 , Z 2, 1 calcd 1.410 g cm À3 , m 1.128 mm À1 , F(000) 2556, 2q max 50.08. Final residuals (for 265 parameters) were R1 0.0491 for 2112 reflections with I > 2sI, and R1 0.0764, wR2 0.1577, GoF 0.985 for all 3520 data. Residual electron density was 0.753 and À 0.574 e ä À3 . Highly disordered solvent molecules in the hexagonal channels were modeled as a group of variable-occupancy carbon atoms. CCDC-165791 contains the supplementary crystallographic

Combined Inhibitor Free‐Energy Landscape and Structural Analysis Reports on the Mannosidase Conformational Coordinate

“…A 1 S 5 → B 2,5 ≠ → O S 2 glycosylation conformational itinerary has been proposed for GH26 mannanases on the basis of the transition-state flanking Michaelis complex in a 1 S 5 conformation,12 and the glycosyl–enzyme intermediate in an O S 2 conformation,13 combined with the principle of least nuclear motion. Only the uncomplexed form of a sole GH113 β-mannanase has been solved and no proposals have been advanced for transition-state conformation for this family 14.…”

β-d-Xylosidase from Selenomonas ruminantium: Role of Glutamate 186 in Catalysis Revealed by Site-Directed Mutagenesis, Alternate Substrates, and Active-Site Inhibitor

“…The strategy was first applied to glycoside hydrolases in the case of SXA acting on 4NPX and 4NPA [11]. The energy barrier for conformational inversion of pyranoses (10 kcal/mol) is considerably higher than that of furanoses (3-4 kcal/mol) [17], and distortion of pyranoside substrates from the ground-state chair conformation is well recognized as a major task for the catalyst [22][23][24][25][26][27][28][29][30]. SXA active-site residues R290 and F31, important for promoting both 4NPA and 4NPX reactions, were identified as being more important for the SXA-catalyzed hydrolysis of 4NPX than that of 4NPA, strongly suggesting involvement of the two residues in promoting conformational inversion.…”

Substrate Distortion by a Lichenase Highlights the Different Conformational Itineraries Harnessed by Related Glycoside Hydrolases

“…The Michaelis complex of this enzyme with unhydrolyzed methylumbelliferyl β‐laminaribioside 1 reveals a conformation between 1 S 3 (skew) and 1,4 B , whereas the covalent reaction intermediate, trapped through the use of the 1,2‐difluoro trisaccharide 2 , is found in the 4 C 1 (chair) conformation. These conformations are in stark contrast to the 1 S 5 and o S 2 conformations observed for the equivalent complexes of the closely related β‐mannanases from this family 4. These observations strongly suggest that individual, even closely related, enzymes harness different conformational itineraries that are necessarily a composite both of enzyme structure and glycoside chemistry.…”

“…Catalysis is thus likely to occur through a simple “electrophilic migration”11 of the anomeric carbon along the reaction coordinate. Notably, however, the 1 S 3 → 4 H 3 → 4 C 1 migration for the family GH26 lichenase is not observed in β‐mannoside hydrolysis by β‐mannanase Cj Man26A (β‐mannanase is the predominant activity displayed by GH26 enzymes) from this family 4…”

“…A central question is raised by these conformational pathways: is the (on‐enzyme) transition‐state structure dictated solely by the chemistry of the parent glycoside, the topography of the enzyme active centre, or a subtle interplay of both? The glycoside hydrolase family GH26 provides a powerful system to investigate these issues as, although most of its members are β‐mannanases,4 it has recently been shown that select GH26 enzymes are specific for “gluco‐configured” substrates, notably β‐1,3:β‐1,4 mixed‐linkage β‐glucan lichenan5 and β‐1,3 linked xylan 6. Herein we present a series of enzymatic snapshots along the reaction coordinate of a family GH26 lichenase from Clostridium thermocellum , hereafter named Ct Lic26A.…”