Three‐dimensional structures of three engineered cellulose‐binding domains of cellobiohydrolase I from <i>Trichoderma reesei</i>

Mattinen, Maija‐Liisa; Kontteli, Maarit; Kerovuo, Janne; Linder, Markus B.; Annila, Arto; Lindeberg, Gunnar; Reinikainen, Tapani; Drakenberg, Torbjörn

doi:10.1002/pro.5560060204

Cited by 80 publications

(71 citation statements)

References 54 publications

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“…position 224 [3]. The pH range (pH [6][7][8] where the change in fluorescence intensity is observed, fits well also with the pK a value of a histidine residue.…”

Section: Fig 2 Melting Temperatures (T M ) and Enzymatic Activity Osupporting

confidence: 70%

The relationship between thermal stability and pH optimum studied with wild‐type and mutant Trichoderma reesei cellobiohydrolase Cel7A

Boer

Koivula

2003

European Journal of Biochemistry

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The major cellulase secreted by the filamentous fungus Trichoderma reesei is cellobiohydrolase Cel7A. Its threedimensional structure has been solved and various mutant enzymes produced. In order to study the potential use of T. reesei Cel7A in the alkaline pH range, the thermal stability of Cel7A was studied as a function of pH with the wildtype and two mutant enzymes using different spectroscopic methods. Tryptophan fluorescence and CD measurements of the wild-type enzyme show an optimal thermostability between pH 3.5-5.6 (T m , 62 ± 2°C), at which the highest enzymatic activity is also observed, and a gradual decrease in the stability at more alkaline pH values. A soluble substrate, cellotetraose, was shown to stabilize the protein fold both at optimal and alkaline pH. In addition, unfolding of the Cel7A enzyme and the release of the substrate seem to coincide at both acidic and alkaline pH, demonstrated by a change in the fluorescence emission maximum. CD measurements were used to show that the five point mutations (E223S/A224H/L225V/T226A/D262G) that together result in a more alkaline pH optimum [Becker, D., Braet, C., Brumer, H., III, Claeyssens, M., Divne, C., Fagerstro¨m, R.B., Harris, M., Jones, T.A., Kleywegt, G.J., Koivula, A., et al. (2001) Biochem. J. 356, 19-30], destabilize the protein fold both at acidic and alkaline pH when compared with the wild-type enzyme. In addition, an interesting time-dependent fluorescence change, which was not observed by CD, was detected for the pH mutant. Our data show that in order to engineer more alkaline pH cellulases, a combination of mutations should be found, which both shift the pH optimum and at the same time improve the thermal stability at alkaline pH range.

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“…position 224 [3]. The pH range (pH [6][7][8] where the change in fluorescence intensity is observed, fits well also with the pK a value of a histidine residue.…”

Section: Fig 2 Melting Temperatures (T M ) and Enzymatic Activity Osupporting

confidence: 70%

The relationship between thermal stability and pH optimum studied with wild‐type and mutant Trichoderma reesei cellobiohydrolase Cel7A

Boer

Koivula

2003

European Journal of Biochemistry

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“…The fusion proteins were constructed by connecting the hydrophobins over the linker region (P263 to P287) of cellobiohydrolase I from T. reesei (GenBank accession no. P62694.1 [18]) to Thc_Cut1 from Thermobifida cellulosilytica (GenBank accession no. ADV92526.1 [8]).…”

Section: Methodsmentioning

confidence: 99%

Enhanced Cutinase-Catalyzed Hydrolysis of Polyethylene Terephthalate by Covalent Fusion to Hydrophobins

Ribitsch

Acero

Przylucka

et al. 2015

Appl Environ Microbiol

164

102

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g Cutinases have shown potential for hydrolysis of the recalcitrant synthetic polymer polyethylene terephthalate (PET). We have shown previously that the rate of this hydrolysis can be enhanced by the addition of hydrophobins, small fungal proteins that can alter the physicochemical properties of surfaces. Here we have investigated whether the PET-hydrolyzing activity of a bacterial cutinase from Thermobifida cellulosilytica (Thc_Cut1) would be further enhanced by fusion to one of three Trichoderma hydrophobins, i.e., the class II hydrophobins HFB4 and HFB7 and the pseudo-class I hydrophobin HFB9b. The fusion enzymes exhibited decreased k cat values on soluble substrates (p-nitrophenyl acetate and p-nitrophenyl butyrate) and strongly decreased the hydrophilicity of glass but caused only small changes in the hydrophobicity of PET. When the enzyme was fused to HFB4 or HFB7, the hydrolysis of PET was enhanced >16-fold over the level with the free enzyme, while a mixture of the enzyme and the hydrophobins led only to a 4-fold increase at most. Fusion with the non-class II hydrophobin HFB9b did not increase the rate of hydrolysis over that of the enzyme-hydrophobin mixture, but HFB9b performed best when PET was preincubated with the hydrophobins before enzyme treatment. The pattern of hydrolysis by the fusion enzymes differed from that of Thc_Cut1 as the concentration of the product mono(2-hydroxyethyl) terephthalate relative to that of the main product, terephthalic acid, increased. Small-angle X-ray scattering (SAXS) analysis revealed an increased scattering contrast of the fusion proteins over that of the free proteins, suggesting a change in conformation or enhanced protein aggregation. Our data show that the level of hydrolysis of PET by cutinase can be significantly increased by fusion to hydrophobins. The data further suggest that this likely involves binding of the hydrophobins to the cutinase and changes in the conformation of its active center. P olyethylene terephthalate (PET) is the best-known and most widespread synthetic polyester in the world. It is used for foils and bottles as well as for fibers for the textile industry (1). Recycling of PET and modification of its properties for different applications by traditional procedures involve harsh chemical and physicochemical treatments (2, 3). Enzymatic modification, particularly by cutinases, has been recognized as a powerful alternative in the past decade (4, 5) and, besides offering new avenues for PET recycling, has the additional advantage of creating a modified PET with increased dyeing efficacy and improved binding to polyvinyl chloride without altering the polymer's bulk properties (4, 5).On the other hand, enzymatic hydrolysis of PET has the inherent disadvantage of occurring at a very low rate (5, 6). The reasons for this are not yet clearly understood. The access of the active center of the cutinase to the insoluble substrate is apparently one of the rate-limiting points, because enlarging the areas around the active sites of cutinases from Fus...

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“…These three aromatic residues are assumed to be involved in the chitin binding of ChiA based on the analogy to the cellulose-binding domains of several cellulases. It is well known that three aromatic residues linearly aligned on the surface of cellulose-binding domains play major roles in cellulose binding (27)(28)(29)(30). In addition, when the 3D structure of CatD ChiA1 complexed with (GlcNAc) 7 was superimposed on the structure of Serratia ChiA, two exposed aromatic residues, Phe-232 and Trp-245, corresponding to Trp-122 and Trp-134 of B. circulans CatD ChiA1 (Fig.…”

Section: Four Aromatic Residues Linearly Aligned On the Surface Ofmentioning

confidence: 99%

Roles of the Exposed Aromatic Residues in Crystalline Chitin Hydrolysis by Chitinase A from Serratia marcescens2170

Uchiyama¹,

Katouno²,

Nikaidou³

et al. 2001

Journal of Biological Chemistry

166

152

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Four exposed aromatic residues, two in the N-terminal domain (Trp-69 and Trp-33) and two in the catalytic domain (Trp-245 and Phe-232) of Serratia marcescens chitinase A, are linearly aligned with the deep catalytic cleft. To investigate the importance of these residues in the binding activity and hydrolyzing activity against insoluble chitin, site-directed mutagenesis to alanine was carried out. The substitution of Trp-69, Trp-33, or Trp-245 significantly reduced the binding activity to both highly crystalline ␤-chitin and colloidal chitin. The substitution of Phe-232, which is located closest to the catalytic cleft, did not affect the binding activity. On the other hand, the hydrolyzing activity against ␤-chitin microfibrils was significantly reduced by the substitution of any one of the four aromatic residues including Phe-232. None of the mutations reduced the hydrolyzing activity against soluble substrates. These results clearly demonstrate that the four exposed aromatic residues are essential determinants for crystalline chitin hydrolysis. Three of them, two in the N-terminal domain and one in the catalytic domain, play vital roles in the chitin binding. Phe-232 appeared to be important for guiding the chitin chain into the catalytic cleft. Based on these observations, a model for processive hydrolysis of crystalline chitin by chitinase A is proposed.

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Three‐dimensional structures of three engineered cellulose‐binding domains of cellobiohydrolase I from Trichoderma reesei

Cited by 80 publications

References 54 publications

The relationship between thermal stability and pH optimum studied with wild‐type and mutant Trichoderma reesei cellobiohydrolase Cel7A

The relationship between thermal stability and pH optimum studied with wild‐type and mutant Trichoderma reesei cellobiohydrolase Cel7A

Enhanced Cutinase-Catalyzed Hydrolysis of Polyethylene Terephthalate by Covalent Fusion to Hydrophobins

Roles of the Exposed Aromatic Residues in Crystalline Chitin Hydrolysis by Chitinase A from Serratia marcescens2170

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