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Multi-mode binding of Cellobiohydrolase Cel7A from Trichoderma reesei to cellulose.

ABSTRACT: Enzymatic hydrolysis of recalcitrant polysaccharides like cellulose takes place on the solid-liquid interface. Therefore the adsorption of enzymes to the solid surface is a pre-requisite for catalysis. Here we used enzymatic activity measurements with fluorescent model-substrate 4-methyl-umbelliferyl-?-D-lactoside for sensitive monitoring of the binding of cellobiohydrolase TrCel7A from Trichoderma reesei to bacterial cellulose (BC). The binding at low nanomolar free TrCel7A concentrations was exclusively active site mediated and was consistent with Langmuir's one binding site model with Kd and Amax values of 2.9 nM and 126 nmol/g BC, respectively. This is the strongest binding observed with non-complexed cellulases and apparently represents the productive binding of TrCel7A to cellulose chain ends on the hydrophobic face of BC microfibril. With increasing free TrCel7A concentrations the isotherm gradually deviated from the Langmuir's one binding site model. This was caused by the increasing contribution of lower affinity binding modes that included both active site mediated binding and non-productive binding with active site free from cellulose chain. The binding of TrCel7A to BC was found to be only partially reversible. Furthermore, the isotherm was dependent on the concentration of BC with more efficient binding observed at lower BC concentrations. The phenomenon can be ascribed to the BC concentration dependent aggregation of BC microfibrils with concomitant reduction of specific surface area.

SUBMITTER: Jalak J

PROVIDER: S-EPMC4180464 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Multi-mode binding of Cellobiohydrolase Cel7A from Trichoderma reesei to cellulose.

Jalak Jürgen J Väljamäe Priit P

PloS one 20140929 9

Enzymatic hydrolysis of recalcitrant polysaccharides like cellulose takes place on the solid-liquid interface. Therefore the adsorption of enzymes to the solid surface is a pre-requisite for catalysis. Here we used enzymatic activity measurements with fluorescent model-substrate 4-methyl-umbelliferyl-β-D-lactoside for sensitive monitoring of the binding of cellobiohydrolase TrCel7A from Trichoderma reesei to bacterial cellulose (BC). The binding at low nanomolar free TrCel7A concentrations was e ...[more]

PMID: 25265511

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Project description:BackgroundCellobiohydrolase from glycoside hydrolase family 7 is a major component of commercial enzymatic mixtures for lignocellulosic biomass degradation. For many years, Trichoderma reesei Cel7A (TrCel7A) has served as a model to understand structure-function relationships of processive cellobiohydrolases. The architecture of TrCel7A includes an N-glycosylated catalytic domain, which is connected to a carbohydrate-binding module through a flexible, O-glycosylated linker. Depending on the fungal expression host, glycosylation can vary not only in glycoforms, but also in site occupancy, leading to a complex pattern of glycans, which can affect the enzyme's stability and kinetics.ResultsTwo expression hosts, Aspergillus oryzae and Trichoderma reesei, were utilized to successfully express wild-types TrCel7A (WT Ao and WT Tr ) and the triple N-glycosylation site deficient mutants TrCel7A N45Q, N270Q, N384Q (ΔN-glyc Ao and ΔN-glyc Tr ). Also, we expressed single N-glycosylation site deficient mutants TrCel7A (N45Q Ao , N270Q Ao , N384Q Ao ). The TrCel7A enzymes were studied by steady-state kinetics under both substrate- and enzyme-saturating conditions using different cellulosic substrates. The Michaelis constant (K M ) was consistently found to be lowered for the variants with reduced N-glycosylation content, and for the triple deficient mutants, it was less than half of the WTs' value on some substrates. The ability of the enzyme to combine productively with sites on the cellulose surface followed a similar pattern on all tested substrates. Thus, site density (number of sites per gram cellulose) was 30-60% higher for the single deficient variants compared to the WT, and about twofold larger for the triple deficient enzyme. Molecular dynamic simulation of the N-glycan mutants TrCel7A revealed higher number of contacts between CD and cellulose crystal upon removal of glycans at position N45 and N384.ConclusionsThe kinetic changes of TrCel7A imposed by removal of N-linked glycans reflected modifications of substrate accessibility. The presence of N-glycans with extended structures increased K M and decreased attack site density of TrCel7A likely due to steric hindrance effect and distance between the enzyme and the cellulose surface, preventing the enzyme from achieving optimal conformation. This knowledge could be applied to modify enzyme glycosylation to engineer enzyme with higher activity on the insoluble substrates.

Project description:BACKGROUND: Mutation of Trp-40 in the Cel7A cellobiohydrolase from Trichoderma reesei (TrCel7A) causes a loss of crystalline cellulose-degrading ability. RESULTS: Mutant W40A showed reduced specific activity for crystalline cellulose and diffused the cellulose chain from the entrance of the active site tunnel. CONCLUSION: Trp-40 is essential for chain end loading to initiate processive hydrolysis of TrCel7A. SIGNIFICANCE: The mechanisms of crystalline polysaccharide degradation are clarified. The glycoside hydrolase family 7 cellobiohydrolase Cel7A from Trichoderma reesei is one of the best studied cellulases with the ability to degrade highly crystalline cellulose. The catalytic domain and the cellulose-binding domain (CBD) are both necessary for full activity on crystalline substrates. Our previous high-speed atomic force microscopy studies showed that mutation of Trp-40 at the entrance of the catalytic tunnel drastically decreases the ability to degrade crystalline cellulose. Here, we examined the activities of the WT enzyme and mutant W40A (with and without the CBD) for various substrates. Evaluation and comparison of the specific activities of the enzymes (WT, W40A, and the corresponding catalytic subunits (WTcat and W40Acat)) adsorbed on crystalline cellulose indicated that Trp-40 is involved in recruiting individual substrate chains into the active site tunnel to initiate processive hydrolysis. This was supported by molecular dynamics simulation study, i.e. the reducing end glucose unit was effectively loaded into the active site of WTcat, but not into that of W40Acat, when the simulation was started from subsite -7. However, when similar simulations were carried out starting from subsite -5, both enzymes held the substrate for 50 ns, indicating that the major difference between WTcat and W40Acat is the length of the free chain end of the substrate required to allow initiation of processive movements; this also reflects the difference between crystalline and amorphous celluloses. The CBD is important for enhancing the enzyme population on crystalline substrate, but it also decreases the specific activity of the adsorbed enzyme, possibly by attaching the enzyme to non-optimal places on the cellulose surface and/or hindering processive hydrolysis.

Dataset Information

Multi-mode binding of Cellobiohydrolase Cel7A from Trichoderma reesei to cellulose.

Publications

Multi-mode binding of Cellobiohydrolase Cel7A from Trichoderma reesei to cellulose.

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