Structural Basis for Ligand Binding and Processivity in Cellobiohydrolase Cel6A from Humicola insolens

Varrot, A.; Frandsen, Torben P.; Ossowski, Ingemar von; Boyer, Viviane; Cottaz, Sylvain; Driguez, Hugues; Schülein, M.; Davies, G.J.

doi:10.1016/s0969-2126(03)00124-2

Cited by 120 publications

(133 citation statements)

References 43 publications

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“…The active site tunnel of TrCel7A is surrounded by four loops with the major one known as an "exo-loop" that roofs the tunnel (9,10,12). Although for family 6 CBHs the mobility of active site loops that allows occasional opening of the tunnel and endo-mode of initiation is well documented (42,43), it has not been demonstrated for family 7 CBHs. Results presented here suggest this possibility also for family 7 CBHs.…”

Section: Measurement Of Cellulase Processivity; Methodsmentioning

confidence: 99%

Processivity of Cellobiohydrolases Is Limited by the Substrate

Kurašin

Väljamaë

2011

Journal of Biological Chemistry

251

350

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Processive cellobiohydrolases (CBHs) are the key components of fungal cellulase systems. Despite the wealth of structural data confirming the processive mode of action, little quantitative information on the processivity of CBHs is available. Here, we developed a method for measuring cellulase processivity. Sensitive fluorescence detection of enzyme-generated insoluble reducing groups on cellulose after labeling with diaminopyridine enabled quantification of the number of reducing-end exo-mode and endo-mode initiations. Both CBHs TrCel7A from Trichoderma reesei and PcCel7D from Phanerochaete chrysosporium employed reducing-end exoand endo-mode initiation in parallel. Processivity values measured for TrCel7A and PcCel7D on cellulose hydrolysis were more than an order of magnitude lower than the values of intrinsic processivity that were found from the ratio of catalytic constant (k cat ) and dissociation rate constant (k off ). We propose that the length of the obstacle-free path available for a processive run on cellulose chain limits the processivity of CBHs on cellulose. TrCel7A and PcCel7D differed in their k off values, whereas the k cat values were similar. Furthermore, the k off values for endoglucanases (EGs) were much higher than the k off values for CBHs, whereas the k cat values for EGs and CBHs were within the same order of magnitude. These results suggest that the value of k off may be the primary target for the selection of cellulases.

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Section: Measurement Of Cellulase Processivity; Methodsmentioning

confidence: 99%

Processivity of Cellobiohydrolases Is Limited by the Substrate

Kurašin

Väljamaë

2011

Journal of Biological Chemistry

251

350

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“…Though the processive mechanism of GHs has been extensively studied by both biochemical and structural studies [8,13**,14**, 18,30,32,33,37,39,[41][42][43][44][45][46][47][48][49][50], the direct connection of structure to processive function beyond the broad topological categorizations remains unclear. We recently described the hypothesized elementary steps involved in the catalytic cycle of a processive GH as a free energy profile along the enzymatic reaction coordinate [51].…”

Section: Thermodynamics Of Cbh Processivitymentioning

confidence: 99%

Towards a molecular-level theory of carbohydrate processivity in glycoside hydrolases

Beckham

Ståhlberg

Knott

et al. 2014

Current Opinion in Biotechnology

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Polysaccharide depolymerization in nature is primarily accomplished by processive glycoside hydrolases (GHs), which abstract single carbohydrate chains from polymer crystals and cleave glycosidic linkages without dissociating after each catalytic event.Understanding the molecular-level features and structural aspects of processivity is of importance due to the prevalence of processive GHs in biomass-degrading enzyme cocktails. Here, we describe recent advances towards the development of a molecular-level theory of processivity for cellulolytic and chitinolytic enzymes, including the development of novel methods for measuring rates of key steps in processive action and insights gained from structural and computational studies. Overall, we present a framework for developing structure-function relationships in processive GHs and outline additional progress towards developing a fundamental understanding of these industrially important enzymes. IntroductionStructural polysaccharides, such as cellulose and chitin, typically arrange in insoluble, polymeric crystals that form significant components of plant, fungal, and algal cell walls. Microorganisms have evolved suites of enzymatic machinery to degrade these polysaccharides to soluble units for food and energy. These enzyme cocktails are primarily composed of various glycoside hydrolases (GHs) with synergistic functions to efficiently cleave the glycosidic linkages [1,2]. More recently, additional enzymatic functions beyond the canonical GH enzyme battery have been discovered including oxidative enzymes that selectively cleave glycosidic bonds [3][4][5][6][7]. GH cocktails contain enzymes typically delineated into two broadly defined classes: cellobiohydrolases (CBHs) and endoglucanases (EGs) for cellulose depolymerization, or chitobiohydrolases and endochitinases for chitin depolymerization. EGs are thought to randomly hydrolyze glycosidic linkages primarily in amorphous regions of polymer fibers. Alternatively, CBHs are able to attach to carbohydrate chains and processively hydrolyze disaccharide units from the end of a chain without dissociation after each catalytic event. Processivity is traditionally thought to be a means of conserving energy during enzymatic function, and is a general strategy used in the synthesis, modification, and depolymerization of many natural biopolymers [8]. It is this ability to act processively that imparts significant hydrolytic potential to CBHs from various GH families such as GH Family 6, 7, 18, and 48 and typically makes them the most abundant enzymes in natural secretomes of many microorganisms. Thus, GHs are the focus of intense protein engineering efforts for the biofuels industry [9*,10].

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“…The substrate-binding sites in processive chitinases and cellulases are lined with aromatic residues, such as tryptophan. These residues are thought to facilitate processivity by functioning as a flexible and hydrophobic sheath along which the polymer chain can slide during the processive mode of action (8,44). Processivity improves the catalytic efficiency of chitinases because the enzyme remains closely associated with the detached single polymer chain between hydrolytic steps (15).…”

mentioning

confidence: 99%

Synthesis of Long-Chain Chitooligosaccharides by a Hypertransglycosylating Processive Endochitinase of Serratia proteamaculans 568

Purushotham

Podile

2012

J Bacteriol

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We describe the heterologous expression and characterization of a 407-residue single-domain glycosyl hydrolase family 18 chitinase (SpChiD) from Gram-negative Serratia proteamaculans 568 that has unprecedented catalytic properties. SpChiD was optimally active at pH 6.0 and 40°C, where it showed a K m of 83 mg ml ؊1 , a k cat of 3.9 ؋ 10 2 h ؊1 , and a k cat /K m of 4.7 h mg ؊1 ml ؊1 on colloidal chitin. On chitobiose, the K m , k cat , and k cat /K m were 203 M, 1.3 ؋ 10 2 h ؊1 , and 0.62 h ؊1 M ؊1 , respectively. Hydrolytic activity on chitooligosaccharides (CHOS) and colloidal chitin indicated that SpChiD was an endo-acting processive enzyme, with the unique ability to convert released chitobiose to N-acetylglucosamine, the major end product. SpChiD showed hyper transglycosylation (TG) with trimer-hexamer CHOS substrates, generating considerable amounts of long-chain CHOS. The TG activity of SpChiD was dependent on both the length and concentration of the oligomeric substrate and also on the enzyme concentration. The length and amount of accumulated TG products increased with increases in the length of the substrate and its concentration and decreased with increases in the enzyme concentration. The SpChiD bound to insoluble and soluble chitin substrates despite the absence of accessory domains. Sequence alignments and structural modeling indicated that SpChiD would have a deep substrate-binding groove lined with aromatic residues, which is characteristic of processive enzymes. SpChiD shows a combination of properties that seems rare among family 18 chitinases and that may resemble the properties of human chitotriosidase.

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Structural Basis for Ligand Binding and Processivity in Cellobiohydrolase Cel6A from Humicola insolens

Cited by 120 publications

References 43 publications

Processivity of Cellobiohydrolases Is Limited by the Substrate

Processivity of Cellobiohydrolases Is Limited by the Substrate

Towards a molecular-level theory of carbohydrate processivity in glycoside hydrolases

Synthesis of Long-Chain Chitooligosaccharides by a Hypertransglycosylating Processive Endochitinase of Serratia proteamaculans 568

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