High temperatures enhance cooperative motions between CBM and catalytic domains of a thermostable cellulase: mechanism insights from essential dynamics

Batista, Paulo Ricardo; Costa, Maurício G. S.; Pascutti, Pedro Geraldo; Bisch, Paulo Mascarello; Souza, Wanderley de

doi:10.1039/c0cp02697b

Cited by 44 publications

(39 citation statements)

References 63 publications

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“…Man5B and Man5A, two glycoside hydrolase family 5 (GH5) enzymes from the same bacterium, were shown to act synergistically and at high temperature on enzymatic conversion of plant cell wall polysaccharides to fermentable sugars [5,6], a property that is highly desirable in the emerging biofuel industry [5,7,8]. …”

Section: Introductionmentioning

confidence: 99%

Molecular dynamics study of enhanced Man5B enzymatic activity

Bernardi

Cann

Schulten

2014

Biotechnol Biofuels

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BackgroundBiofuels are a well-known alternative to the largely used fossil-derived fuels, however the competition with food production is an ethical dilemma. Fortunately a solution is offered by second-generation biofuels which can be produced from agricultural waste or, more specifically, from plant cell wall polysaccharides. The conversion process involves typically enzymatic hydrolysis of lignocellulosic biomass and then separation of its constituent sugars that are further fermented to produce ethanol. Over the years several technologies have been developed that allow this conversion process to occur and the objective is now to make this process cost-competitive in today’s markets.ResultsWe observe that reduction of enzymatic efficiency in the presence of gluco-oligosaccharides is associated with a loss of the enzyme’s flexibility, the latter being required to bind new substrate, while the presence of manno-oligosaccharides does not pose this problem. Molecular dynamics simulations identify key contacts between substrates and the enzyme catalytic pocket that might be modified through site-directed mutagenesis to prevent loss of enzymatic efficiency.ConclusionsBased on previous experimental studies and the new molecular dynamics data, we suggest that cellohexaose in the active site pocket slows down or even inhibits Man5B enzymatic activity. The assumption of such a mechanism is reasonable since when the gluco-oligosaccharide substrate is attached to the catalytic pocket it takes much longer to leave the pocket and thus prevents other substrates from reaching the active site. The insight is of crucial importance since the inhibition of enzymes by the enzymatic product or by an unsuitable substrate is a major technological problem in reducing the competitiveness of second-generation biofuel production.

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Section: Introductionmentioning

confidence: 99%

Molecular dynamics study of enhanced Man5B enzymatic activity

Bernardi

Cann

Schulten

2014

Biotechnol Biofuels

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“…Unlike PcCel45A, EG II and EG II-E have a higher specific activity on PASC than on CMC, which might be caused by the different structures. CBM domains are important to recognize, coact and boost the incorporation of the cellulase with the substrate (Batista et al 2011). The S39 of CBM1 was reported to participate in forming the hydrophilic surface of cellulase crucial for carbohydrate binding as described previously (Kraulis et al 1989).…”

Section: Discussionmentioning

confidence: 93%

“…Generally, there are three components of endoglucanase, the cellulose-binding domain (CBD), the catalytic domain (CD) and a linker with different length (Béguin and Aubert 1994). CBD is composed of various carbohydrate-binding modules (CBM) which are very essential for the recognition and incorporation of the cellulase with substrates (Batista et al 2011). CD is substrate-specific, and can play a catalytic role independently (Warner et al 2013).…”

Section: Introductionmentioning

confidence: 99%

Expression and functional analysis of a glycoside hydrolase family 45 endoglucanase from Rhizopus stolonifer

Tang

Zhang

Yang

et al. 2014

World J Microbiol Biotechnol

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A novel endoglucanase gene was cloned from Rhizopus stolonifer and expressed in Escherichia coli, the gene product EG II (45 kDa) was assigned to Glycoside Hydrolase Family 45 (GH45), and its specific activity on phosphoric acid-swollen cellulose (PASC) was 48 IU/mg. To solve the problem of substrate accumulation in the cellulose hydrolysis and enhance the catalytic efficiency of endoglucanase, the eg2 gene was modified by site directed mutagenesis. Mutations generated by overlapping PCR have been proven to increase its catalytic activity on carboxymenthyl cellulose, microcrystalline cellulose (Avicel) and PASC, among which the mutant EG II-E containing all 6 mutations (N39S, V136D, T251G, D255G, P256S and E260D) peaked 121 IU/mg on PASC. The bioinformatic analysis showed that 2 key catalytic residues (D136 and D260) moved closer with the opening of a loop after mutagenesis, and a tunnel was formed by structural transformation. This structure was conducive for the substrate to access the active centre, and D136 played an indispensable role in the substrate recognition.

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“…Reports show that linker length and stiffness play a critical role in the cooperative action of cellulase domains [28]. The hinge bending motion of the semi-stiff linker is beneficial for the synergistic effect of the cellulase domains [28,29]. However, quantification of the relationship between linker length and stiffness of the PT-box is challenging.…”

Section: Mutant Design Of Accel12b Linkermentioning

confidence: 99%

“…Thus, alanine may act as a hinge that renders semi-flexibility to the otherwise-stiff PT/S-box polypeptide chain. Reports show that linker length and stiffness play a critical role in the cooperative action of cellulase domains [28]. The hinge bending motion of the semi-stiff linker is beneficial for the synergistic effect of the cellulase domains [28,29].…”

Section: Mutant Design Of Accel12b Linkermentioning

confidence: 99%

The PT/S-Box of Modular Cellulase AcCel12B Plays a Key Role in the Hydrolysis of Insoluble Cellulose

Wang

et al. 2018

Catalysts

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Abstract:Cellulases play key roles in the degradation of lignocellulosic materials. The function and mechanism of the catalytic domain (CD) and carbohydrate-binding module (CBM) of cellulases were earlier revealed by analysis and characterization of protein structure. However, understanding of the catalytic mechanism of the entire enzyme, and the analysis of the catalytic model, were inadequate. Therefore, the linker chain between CD and CBM has been extensively studied to bridge this gap. Cellulase AcCel12B and three mutants with different linker lengths (with no or 1-3 PT/S-box units) were successfully constructed and purified. Results showed that the activity of cellulases on Avicel and regenerated amorphous cellulose (RAC) increased with the number of PT/S-box units. Furthermore, the desorption of AcCel12B and its mutants from RAC and Avicel were significantly different. The energy of desorption of wild-type and mutant AcCel12B from cellulose decreased with the number of PT/S-box units. Thus, AcCel12B containing more PT/S-box units was more easily desorbed and had more opportunity to hydrolyze cellulose than other samples. The number of PT/S-box units in endocellulase affected the desorption of the enzyme, which is possibly responsible for the differences in the activity of wild-type and mutant AcCel12B on Avicel and RAC.

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High temperatures enhance cooperative motions between CBM and catalytic domains of a thermostable cellulase: mechanism insights from essential dynamics

Cited by 44 publications

References 63 publications

Molecular dynamics study of enhanced Man5B enzymatic activity

Molecular dynamics study of enhanced Man5B enzymatic activity

Expression and functional analysis of a glycoside hydrolase family 45 endoglucanase from Rhizopus stolonifer

The PT/S-Box of Modular Cellulase AcCel12B Plays a Key Role in the Hydrolysis of Insoluble Cellulose

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