How Family 26 Glycoside Hydrolases Orchestrate Catalysis on Different Polysaccharides

Taylor, Edward J.; Goyal, Arun; Guerreiro, Catarina I. P. D.; Prates, José A. M.; Money, Victoria A.; Ferry, Nicolas; Morland, Carl; Planas, Antoni; Macdonald, J. A.; Stick, Robert V.; Gilbert, Harry J.; Fontes, Carlos M. G. A.; Davies, G.J.

doi:10.1074/jbc.m506580200

Cited by 61 publications

(41 citation statements)

References 43 publications

(36 reference statements)

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“…[3] 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 b-mannanases, [4] it has recently been shown that select GH26 enzymes are specific for "gluco-configured" substrates, notably b-1,3:b-1,4 mixedlinkage b-glucan lichenan [5] and b-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 CtLic26A.…”

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“…[5] Catalysis occurs with net retention of the anomeric configuration through the formation (glycosylation) and subsequent breakdown (deglycosylation) of a covalent glycosyl-enzyme intermediate that is flanked on either side by oxocarbenium-ion-like transition states. Such a reaction demands two essential catalytic groups: a nucleophile and an acid/base that, in this family, are both enzyme-derived carboxylates.…”

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

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

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“…Such analyses should provide a biological rationale for the repertoire of glycoside hydrolases produced by both prokaryotic and eukaryotic systems. Indeed, until recently, GH26 was thought to be exclusively an endo-␤-1,4-mannanase family, but two members that display, respectively, ␤-1,3-xylanase (12) and ␤-1,4:␤-1,3-glucanase activities (13,14) have now been described. This diversity in function is further exemplified by the recent identification of a GH5 enzyme, CmMan5A, which, although displaying extensive sequence identity to endo-acting mannanases, is an exo-acting mannanase that releases mannose from the nonreducing end of polymeric substrates (7).…”

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The Cellvibrio japonicus Mannanase CjMan26C Displays a Unique exo-Mode of Action That Is Conferred by Subtle Changes to the Distal Region of the Active Site

Cartmell

Topakas

Ducros

et al. 2008

Journal of Biological Chemistry

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The microbial degradation of the plant cell wall is a pivotal biological process that is of increasing industrial significance. One of the major plant structural polysaccharides is mannan, a ␤-1,4-linked D-mannose polymer, which is hydrolyzed by endoand exo-acting mannanases. The mechanisms by which the exoacting enzymes target the chain ends of mannan and how galactose decorations influence activity are poorly understood. Here we report the crystal structure and biochemical properties of CjMan26C, a Cellvibrio japonicus GH26 mannanase. The exoacting enzyme releases the disaccharide mannobiose from the nonreducing end of mannan and mannooligosaccharides, harnessing four mannose-binding subsites extending from ؊2 to ؉2. The structure of CjMan26C is very similar to that of the endo-acting C. japonicus mannanase CjMan26A. The exo-activity displayed by CjMan26C, however, reflects a subtle change in surface topography in which a four-residue extension of surface loop creates a steric block at the distal glycone ؊2 subsite. endoActivity can be introduced into enzyme variants through truncation of an aspartate side chain, a component of a surface loop, or by removing both the aspartate and its flanking residues. The structure of catalytically competent CjMan26C, in complex with a decorated manno-oligosaccharide, reveals a predominantly unhydrolyzed substrate in an approximate 1 S 5 conformation. The complex structure helps to explain how the substrate "side chain" decorations greatly reduce the activity of the enzyme; the galactose side chain at the ؊1 subsite makes polar interactions with the aglycone mannose, possibly leading to suboptimal binding and impaired leaving group departure. This report reveals how subtle differences in the loops surrounding the active site of a glycoside hydrolase can lead to a change in the mode of action of the enzyme.The plant cell wall represents the dominant source of organic carbon in the biosphere and as such supports many facets of terrestrial and marine life. Access to this valuable source of carbon is mediated by extensive microbial enzyme consortia, which generate sugars that are utilized by microbial ecosystems to the benefit of plants, mammalian herbivores, and insects. These degradative enzymes are increasingly deployed in industrial processes, and it is apparent that their use in producing second generation, lignocellulosebased, biofuels is of considerable environmental significance (1, 2). Among the major polysaccharides in softwoods and angiosperms are the mannans. These polysaccharides comprise a backbone of ␤-1,4-linked D-mannopyranose sugars (known as "mannan") or a heterogeneous combination of ␤-1,4-D-mannose and ␤-1,4-D-glucose units (termed "glucomannan"), which can be decorated with ␣-1,6-galactose side chains to yield "galactomannan" and "galactoglucomannan," respectively (3). For complete enzymatic degradation, the galactose decorations are removed by ␣-galactosidases (4, 5), whereas the internal glycosidic linkages in mannans are cleaved by endo-␤-1,4 mannanases ...

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“…1). A current issue in the understanding of GH mechanisms is the conformational itinerary that the substrate follows during the reaction (5,6), in which substrate distortion is induced upon binding to the enzyme to reach a transition state with sp 2 geometry at the anomeric carbon.…”

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Substrate Distortion in the Michaelis Complex of Bacillus 1,3–1,4-β-Glucanase

Biarnés

Nieto

Planas

et al. 2006

Journal of Biological Chemistry

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The structure and dynamics of the enzyme-substrate complex of Bacillus 1,3-1,4-␤-glucanase, one of the most active glycoside hydrolases, is investigated by means of Car-Parrinello molecular dynamics simulations (CPMD) combined with force field molecular dynamics (QM/MM CPMD). It is found that the substrate sugar ring located at the ؊1 subsite adopts a distorted 1 S 3 skew-boat conformation upon binding to the enzyme. With respect to the undistorted 4 C 1 chair conformation, the 1 S 3 skew-boat conformation is characterized by: (a) an increase of charge at the anomeric carbon (C1), (b) an increase of the distance between C1 and the leaving group, and (c) a decrease of the intraring O5-C1 distance. Therefore, our results clearly show that the distorted conformation resembles both structurally and electronically the transition state of the reaction in which the substrate acquires oxocarbenium ion character, and the glycosidic bond is partially broken. Together with analysis of the substrate conformational dynamics, it is concluded that the main determinants of substrate distortion have a structural origin. To fit into the binding pocket, it is necessary that the aglycon leaving group is oriented toward the ␤ region, and the skew-boat conformation naturally fulfills this premise. Only when the aglycon is removed from the calculation the substrate recovers the all-chair conformation, in agreement with the recent determination of the enzyme product structure. The QM/MM protocol developed here is able to predict the conformational distortion of substrate binding in glycoside hydrolases because it accounts for polarization and charge reorganization at the ؊1 sugar ring. It thus provides a powerful tool to model E⅐S complexes for which experimental information is not yet available. Glycoside hydrolases (GHs)2 are the enzymes responsible for the hydrolysis of glycosidic bonds and play important biological functions such as glycan processing in glycoproteins, remodeling the cell walls, and polysaccharide modification and degradation. The reaction mechanism, a classical textbook example of enzymatic reaction, has attracted much interest because genetically inherited disorders of glycoside hydrolysis often occur and because inhibitors of these enzymes can act as new therapeutic agents for the treatment of viral infections (1, 2). Despite the large number of GHs known, classified into more than 90 families (1), the catalytic mechanism is similar. They typically operate by means of acid/base catalysis with retention or inversion of the anomeric configuration, although a different mechanism has recently been proposed for the GH family 4 (3). The acid/base reaction is assisted by two essential residues: a proton donor and a nucleophile or general base residue (4). Inverting enzymes operate by a single nucleophilic substitution, whereas retaining glycosidases follow a double displacement mechanism via formation and hydrolysis of a covalent glycosyl-enzyme intermediate. Both steps involve oxocarbenium ion-like transition states (F...

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How Family 26 Glycoside Hydrolases Orchestrate Catalysis on Different Polysaccharides

Cited by 61 publications

References 43 publications

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

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

The Cellvibrio japonicus Mannanase CjMan26C Displays a Unique exo-Mode of Action That Is Conferred by Subtle Changes to the Distal Region of the Active Site

Substrate Distortion in the Michaelis Complex of Bacillus 1,3–1,4-β-Glucanase

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