Activity Studies and Crystal Structures of Catalytically Deficient Mutants of Cellobiohydrolase I fromTrichoderma reesei

Ståhlberg, Jerry; Divne, Christina; Koivula, Anu; Piens, Kathleen; Claeyssens, Marc; Teeri, Tuula T.; Jones, T. Alwyn

doi:10.1006/jmbi.1996.0644

Cited by 162 publications

(230 citation statements)

References 49 publications

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“…The four surface loops forming the substrate-binding tunnel vary only slightly in amino acid sequence, and are identical in length in all four cellobiohydrolases studied here. Sitedirected mutagenesis studies of T. reesei Cel7A have shown that there are three catalytic residues in the active site, where Glu212 acts as the possible nucleophile, Glu217 as a proton donor, and Asp214 is responsible of maintaining the correct positioning and protonation state of Glu212 by the hydrogen bond formed between Asp214 and Glu212 (Divne et al, 1998;Ståhlberg et al, 1996). These three carboxylic acids are conserved in all three thermostable cellobiohydrolases, as shown in the sequence alignment (Fig.…”

Section: Discussionmentioning

confidence: 78%

“…The catalytic site is located at one end of the tunnel and enables product release from the reducing end of the cellulose chain, while the rest of the polymer chain is still bound to the enzyme. Structure-based protein engineering has been applied to study, for example, the details of the catalytic mechanism (Divne et al, 1998), the role of the surface loops and the pH behavior of Tr Cel7A (Becker et al, 2001;Ståhlberg et al, 1996).…”

Section: Introductionmentioning

confidence: 99%

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Cloning, expression, and characterization of novel thermostable family 7 cellobiohydrolases

Voutilainen

Puranen

Siika‐aho

et al. 2008

Biotech & Bioengineering

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111

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As part of the effort to find better cellulases for bioethanol production processes, we were looking for novel GH-7 family cellobiohydrolases, which would be particularly active on insoluble polymeric substrates and participate in the rate-limiting step in the hydrolysis of cellulose. The enzymatic properties were studied and are reported here for family 7 cellobiohydrolases from the thermophilic fungi Acremonium thermophilum, Thermoascus aurantiacus, and Chaetomium thermophilum. The Trichoderma reesei Cel7A enzyme was used as a reference in the experiments. As the native T. aurantiacus Cel7A has no carbohydrate-binding module (CBM), recombinant proteins having the CBM from either the C. thermophilum Cel7A or the T. reesei Cel7A were also constructed. All these novel acidic cellobiohydrolases were more thermostable (by 4-108C) and more active (two-to fourfold) in hydrolysis of microcrystalline cellulose (Avicel) at 458C than T. reesei Cel7A. The C. thermophilum Cel7A showed the highest specific activity and temperature optimum when measured on soluble substrates. The most effective enzyme for Avicel hydrolysis at 708C, however, was the 2-module version of the T. aurantiacus Cel7A, which was also relatively weakly inhibited by cellobiose. These results are discussed from the structural point of view based on the three-dimensional homology models of these enzymes.

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Section: Discussionmentioning

confidence: 78%

Section: Introductionmentioning

confidence: 99%

Cloning, expression, and characterization of novel thermostable family 7 cellobiohydrolases

Voutilainen

Puranen

Siika‐aho

et al. 2008

Biotech & Bioengineering

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111

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“…Neither is there a "rear exit" for the departure of released glucose units from the bottom of the pocket, as there is for the release of the reaction product, cellobiose, through the rear of the tunnel found in the family 6 exohydrolase, cellobiohydrolase II from Trichoderma reesei (37,44,45). In the case of the barley ␤-glucosidase, it seems likely that the substrate is at least partially released from the enzyme after each hydrolytic event, although the oligosaccharide product could remain inserted in the pocket for rapid re-binding prior to the next hydrolytic event.…”

Section: Discussionmentioning

confidence: 99%

Substrate Binding and Catalytic Mechanism of a Barley β-d-Glucosidase/(1,4)-β-d-Glucan Exohydrolase

Hrmová¹,

MacGregor²,

Biely³

et al. 1998

Journal of Biological Chemistry

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A ␤-glucosidase, designated isoenzyme ␤II, from germinated barley (Hordeum vulgare L.) hydrolyzes aryl-␤-glucosides and shares a high level of amino acid sequence similarity with ␤-glucosidases of diverse origin. It releases glucose from the non-reducing termini of cellodextrins with catalytic efficiency factors, k cat /K m , that increase approximately 9-fold as the degree of polymerization of these substrates increases from 2 to 6. Thus, the enzyme has a specificity and action pattern characteristic of both ␤-glucosidases (EC 3.2.1.21) and the polysaccharide exohydrolase, (1,4)-␤-glucan glucohydrolase (EC 3.2.1.74). At high concentrations (100 mM) of 4-nitrophenyl ␤-glucoside, ␤-glucosidase isoenzyme ␤II catalyzes glycosyl transfer reactions, which generate 4-nitrophenyl-␤-laminaribioside, -cellobioside, and -gentiobioside. Subsite mapping with cellooligosaccharides indicates that the barley ␤-glucosidase isoenzyme ␤II has six substrate-binding subsites, each of which binds an individual ␤-glucosyl residue. Amino acid residues Glu 181 and Glu 391 are identified as the probable catalytic acid and catalytic nucleophile, respectively. The enzyme is a family 1 glycoside hydrolase that is likely to adopt a (␤/␣) 8 barrel fold and in which the catalytic amino acid residues appear to be located at the bottom of a funnel-shaped pocket in the enzyme.Two ␤-glucosidases of apparent molecular mass 62,000 have been purified from extracts of germinated barley grain (1-3) and can be classified in the family 1 group of glycosyl hydrolases and related enzymes (4). The two enzymes have been designated isoenzymes ␤I and ␤II, and have isoelectric points of 8.9 and 9.0, respectively. Amino acid sequence analyses reveal a single amino acid difference in the first 50 NH 2 -terminal amino acid residues, and the complete amino acid sequence of isoenzyme ␤II has been deduced from the nucleotide sequence of a corresponding cDNA clone (2).The function of the ␤-glucosidases in the germinated barley grain has not been defined unequivocally, but will clearly be related to their substrate specificities. It has been widely assumed that ␤-glucosidases prefer substrates of the type G-O-X, where G indicates the glucosyl residue and X can either be another glycosyl residue, for which the linkage position is not crucial, or a non-glycosyl aglycone group. As a result of the capacity of many ␤-glucosidases to hydrolyze glucosides with a range of glycosyl or non-glycosyl aglycone groups, non-physiological substrates such as 4-nitrophenyl ␤-D-glucopyranoside (4-NPG) 1 have been synthesized to measure activity in convenient spectrophotometric assays; the barley enzymes have also been assayed in this way. It has further been assumed that the rate of hydrolysis of oligomeric substrates by ␤-glucosidases will remain approximately constant or decrease with increasing degree of polymerization (DP) of the substrate (5). However, the barley ␤-glucosidase isoenzyme ␤II hydrolyzes (1,4)-␤-oligoglucosides much more efficiently than it hydrolyzes the aryl-␤-gluco...

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“…The segment encoding the seventh cohesin domain of CipA, plus a 16-residue linker segment, was cloned from pCip3 (22). The sequence encoding CBD CBH1 with a six-residue linker was cloned from pEMF5 (23). The segment encoding CBD CBH2 and six residues from the adjacent linker was carried by pTTc9 (24).…”

Section: Methodsmentioning

confidence: 99%

Cellulose-binding domains promote hydrolysis of different sites on crystalline cellulose

Carrard

Koivula

Söderlund

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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180

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Activity Studies and Crystal Structures of Catalytically Deficient Mutants of Cellobiohydrolase I fromTrichoderma reesei

Cited by 162 publications

References 49 publications

Cloning, expression, and characterization of novel thermostable family 7 cellobiohydrolases

Cloning, expression, and characterization of novel thermostable family 7 cellobiohydrolases

Substrate Binding and Catalytic Mechanism of a Barley β-d-Glucosidase/(1,4)-β-d-Glucan Exohydrolase

Cellulose-binding domains promote hydrolysis of different sites on crystalline cellulose

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