Glycoside hydrolase family 5: structural snapshots highlighting the involvement of two conserved residues in catalysis

Collet, Laetitia; Wauven, Corinne Vander; Oudjama, Yamina; Galleni, Moreno; Dutoit, Raphaël

doi:10.1107/s2059798320015557

Cited by 3 publications

(16 citation statements)

References 47 publications

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“…Firstly, the size of the acceptor sugar was found to be decisive and must be of at least three sugar units. This implies the existence of a third positive subsite which had not previously been described (Delsaute et al, 2013;Collet et al, 2021). The structure of the Glu135Gln variant of RBcel1 was solved in complex with cellotriose.…”

Section: Introductionmentioning

confidence: 83%

“…6b). Only four negatively numbered subsites have been described so far (Delsaute et al, 2013;Collet et al, 2021). However, the presence of G6 in the negatively numbered subsites of RBcel1 in monomer B allowed the definition of two additional subsites: À5 and À6.…”

Section: Snapshots Of Rbcel1_y201f On the Way To Transglycosylationmentioning

confidence: 99%

“…The original pET-22b-RBcel1 expression vector used for the production of wild-type RBcel1 protein (RBcel1_WT) has been described previously (Berlemont et al, 2009), as well as the pBAD-RBcel1 vector for the production of the RBcel1 variant with Tyr201 substituted with a phenylalanine residue (RBcel1_Y201F; Collet et al, 2021). The RBcel1 variant with Glu135 substituted with a glutamine residue (RBcel1_E135Q) was produced from pET-22b-RBcel1 after targeted mutagenesis with the QuikChange Site-Directed Mutagenesis Kit (Agilent).…”

Section: Cloning and Mutagenesis Of Rbcel1mentioning

confidence: 99%

“…RBcel1, an endoglucanase of the GH5_5 subfamily isolated from an uncultured bacterium, has aroused interest because of its ability to polymerize cellooligosaccharides in vitro under near-physiological conditions (Berlemont et al, 2009). In a recent study, we reported that a single substitution in its active site, Tyr201Phe, stabilizes the formation of the glycosylenzyme covalent intermediate (GEI; Collet et al, 2021). The structure of the GEI obtained with the natural oligosaccharide cellotriose delivered a series of snapshots of the reaction mechanism.…”

Section: Introductionmentioning

confidence: 99%

“…The structure of the GEI obtained with the natural oligosaccharide cellotriose delivered a series of snapshots of the reaction mechanism. Tyr201 is a highly conserved residue among GH5 family members and is believed to play an important role in both glycosylation and deglycosylation (Ducros et al, 1995;Sakon et al, 1996;Collet et al, 2021;Gonc ¸alves et al, 2012;Kim & Ishikawa, 2011;Zheng et al, 2012). The Tyr201Phe variant of RBcel1 retains transglycosylation activity, although it is reduced.…”

Section: Introductionmentioning

confidence: 99%

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Highlighting the factors governing transglycosylation in the GH5_5 endo-1,4-β-glucanase RBcel1

Collet¹,

Wauven²,

Oudjama³

et al. 2022

Acta Cryst Sect D Struct Biol

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Transglycosylating glycoside hydrolases (GHs) offer great potential for the enzymatic synthesis of oligosaccharides. Although knowledge is progressing, there is no unique strategy to improve the transglycosylation yield. Obtaining efficient enzymatic tools for glycan synthesis with GHs remains dependent on an improved understanding of the molecular factors governing the balance between hydrolysis and transglycosylation. This enzymatic and structural study of RBcel1, a transglycosylase from the GH5_5 subfamily isolated from an uncultured bacterium, aims to unravel such factors. The size of the acceptor and donor sugars was found to be critical since transglycosylation is efficient with oligosaccharides at least the size of cellotetraose as the donor and cellotriose as the acceptor. The reaction pH is important in driving the balance between hydrolysis and transglycosylation: hydrolysis is favored at pH values below 8, while transglycosylation becomes the major reaction at basic pH. Solving the structures of two RBcel1 variants, RBcel1_E135Q and RBcel1_Y201F, in complex with ligands has brought to light some of the molecular factors behind transglycosylation. The structure of RBcel1_E135Q in complex with cellotriose allowed a +3 subsite to be defined, in accordance with the requirement for cellotriose as a transglycosylation acceptor. The structure of RBcel1_Y201F has been obtained with several transglycosylation intermediates, providing crystallographic evidence of transglycosylation. The catalytic cleft is filled with (i) donors ranging from cellotriose to cellohexaose in the negative subsites and (ii) cellobiose and cellotriose in the positive subsites. Such a structure is particularly relevant since it is the first structure of a GH5 enzyme in complex with transglycosylation products that has been obtained with neither of the catalytic glutamate residues modified.

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Section: Introductionmentioning

confidence: 83%

Section: Snapshots Of Rbcel1_y201f On the Way To Transglycosylationmentioning

confidence: 99%

Section: Cloning and Mutagenesis Of Rbcel1mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Highlighting the factors governing transglycosylation in the GH5_5 endo-1,4-β-glucanase RBcel1

Collet¹,

Wauven²,

Oudjama³

et al. 2022

Acta Cryst Sect D Struct Biol

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Fungal cellulases: protein engineering and post-translational modifications

Zhang

Cao²,

Bi³

et al. 2021

Appl Microbiol Biotechnol

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The Role of a Loop in the Non-catalytic Domain B on the Hydrolysis/Transglycosylation Specificity of the 4-α-Glucanotransferase from Thermotoga maritima

Llopiz,

Ramírez-Martínez,

Olvera

et al. 2023

Protein J

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The mechanism by which glycoside hydrolases control the reaction specificity through hydrolysis or transglycosylation is a key element embedded in their chemical structures. The determinants of reaction specificity seem to be complex. We looked for structural differences in domain B between the 4-α-glucanotransferase from Thermotoga maritima (TmGTase) and the α-amylase from Thermotoga petrophila (TpAmylase) and found a longer loop in the former that extends towards the active site carrying a W residue at its tip. Based on these differences we constructed the variants W131G and the partial deletion of the loop at residues 120-124/128-131, which showed a 11.6 and 11.4-fold increased hydrolysis/transglycosylation (H/T) ratio relative to WT protein, respectively. These variants had a reduction in the maximum velocity of the transglycosylation reaction, while their affinity for maltose as the acceptor was not substantially affected. Molecular dynamics simulations allow us to rationalize the increase in H/T ratio in terms of the flexibility near the active site and the conformations of the catalytic acid residues and their associated pKas.

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Glycoside hydrolase family 5: structural snapshots highlighting the involvement of two conserved residues in catalysis

Cited by 3 publications

References 47 publications

Highlighting the factors governing transglycosylation in the GH5_5 endo-1,4-β-glucanase RBcel1

Highlighting the factors governing transglycosylation in the GH5_5 endo-1,4-β-glucanase RBcel1

Fungal cellulases: protein engineering and post-translational modifications

The Role of a Loop in the Non-catalytic Domain B on the Hydrolysis/Transglycosylation Specificity of the 4-α-Glucanotransferase from Thermotoga maritima

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