Isolated fungal cellulose terminal domains and a synthetic minimum analogue bind to cellulose

Johansson, Gunnar; Ståhlberg, Jerry; Lindeberg, Gunnar; Engström, Åke; Pettersson, Göran

doi:10.1016/0014-5793(89)80168-1

Cited by 80 publications

(34 citation statements)

References 23 publications

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“…Their binding to crystalline cellulose was determined in order to identify residues responsible for the increased affinity of the EGI CBD. It should be noted that the CBDs used here were made synthetically, and that we have demonstrated earlier, in the case of CBHI that there is no difference in binding between the natural and synthetic CBDs [12].…”

Section: Resultsmentioning

confidence: 74%

The difference in affinity between two fungal cellulose‐binding domains is dominated by a single amino acid substitution

et al. 1995

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Cellulose-binding domains (CBDs) form distinct functional units of most cellulolytic enzymes. We have compared the cellulose-binding affinities of the CBDs of ceilobiohydrolase 1 (CBHI) and endoglucanase I (EGI) from the fungus Trichoderma reeseL The CBD of EGI had significantly higher affinity than that of CBHI. Four variants of the CBHI CBD were made in order to identify the residues responsible for the increased affinity in EGI. Most of the difference could be ascribed to a replacement of a tyrosine by a tryptophan on the flat cellulose-binding face.

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Section: Resultsmentioning

confidence: 74%

The difference in affinity between two fungal cellulose‐binding domains is dominated by a single amino acid substitution

et al. 1995

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“…1 shows the amino acid sequence of the isolated CBHI CBD with the remaining 11 amino acid residues of the linker. This linker segment is not regarded as a part of the CBD and does not affect its binding to cellulose (6,29). Reversed-phase chromatography and MALDI/TOF mass spectroscopy confirmed both the purity and identity of the isolated protein, the calculated mass being 4696.63 Da and the experimentally measured one 4695.34 Da.…”

Section: Methodsmentioning

confidence: 69%

The cellulose-binding domain of the major cellobiohydrolase of Trichoderma reesei exhibits true reversibility and a high exchange rate on crystalline cellulose.

Linder

Teeri

1996

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

165

126

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Cellulose-binding domains (CBDs) bind specifically to cellulose, and form distinct domains of most cellulose degrading enzymes. The CBD-mediated binding of the enzyme has a fundamental role in the hydrolysis of the solid cellulose substrate. In this work we have investigated the reversibility and kinetics of the binding of the CBD from Trichoderma reesei cellobiohydrolase I on microcrystalline cellulose. The CBD was produced in Escherichia coli, purified, and radioactively labeled by reductive alkylation with 3H.Sensitive detection of the labeled CBD allowed more detailed analysis of its behavior than has been possible before, and important novel features were resolved. Binding of the CBD was found to be temperature sensitive, with an increased affinity at lower temperatures. The interaction of the CBD with cellulose was shown to be fully reversible and the CBD could be eluted from cellulose by simple dilution. The rate of exchange measured for the CBD-cellulose interaction compares well with the hydrolysis rate of cellobiohydrolase I, which is consistent with its proposed mode of action as a processive exoglucanase.Cellulose is a polymer composed of D-glucose units linked by 13-1,4-glycosidic bonds. The cellulose chains can pack together to form fibers, which are crystalline in nature (1). This solid and ordered substrate poses some special requirements on the enzymes hydrolyzing it. Most cellulose degrading enzymes have a bifunctional organization composed of Iwo domains connected by a linker region. One of the domaind is dedicated for binding to the solid cellulose and is called a cellulosebinding domain (CBD). The other domain contains the catalytic machinery. These catalytic domains have been grouped into several families sharing the same basic structure and the same chemical reaction mechanism (2). Trichoderma reesei cellobiohydrolase I (CBHI) is an exoglucanase with a long tunnel-shaped active site (3). CBHI and other exoglucanases initiate the hydrolysis at the chain ends, and do not produce significant amounts of new chain ends on the cellulose surface (4). They seem to act by a processive mechanism hydrolyzing consecutive bonds along the same chain without dissociating (2, 3). The endoglucanases cleave internal bonds along the cellulose chains probably by cycles of adsorption and desorption.Based on sequence comparisons the CBDs have been grouped into several families (5). To date, the threedimensional structures of two CBDs have been published. One is from T. reesei CBHI (6) and the other from Cellulomonas fimi xylanase/cellobiohydrolase Cex (7). Both the fold and the size of these CBDs are different, but a common pattern of aromatic residues and amides is found at the surface of both proteins. Many experiments have shown that this surface is the primary interaction site between a CBD and cellulose (7-10). Many studies directly correlate the efficiency of crystalline cellulose degradation to the binding efficiency of the enzyme (11) and the removal of a CBD typically results in a decrease of a...

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“…Importantly, we found that O-mannosylation at Thr1, Ser3, and Ser14 sites does not impair CBM folding. As anticipated, folding of the deprotected glycopeptides can be initiated by direct dilution of the deprotection mixture in a mixed glutathione-folding buffer without additional extraction or purification (31). Properly folded CBM glycoforms display much shorter retention times on HPLC than side products, allowing for facile purification.…”

Section: Resultsmentioning

confidence: 99%

Specificity of O -glycosylation in enhancing the stability and cellulose binding affinity of Family 1 carbohydrate-binding modules

Chen

Drake

Resch

et al. 2014

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

154

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The majority of biological turnover of lignocellulosic biomass in nature is conducted by fungi, which commonly use Family 1 carbohydrate-binding modules (CBMs) for targeting enzymes to cellulose. Family 1 CBMs are glycosylated, but the effects of glycosylation on CBM function remain unknown. Here, the effects of O-mannosylation are examined on the Family 1 CBM from the Trichoderma reesei Family 7 cellobiohydrolase at three glycosylation sites. To enable this work, a procedure to synthesize glycosylated Family 1 CBMs was developed. Subsequently, a library of 20 CBMs was synthesized with mono-, di-, or trisaccharides at each site for comparison of binding affinity, proteolytic stability, and thermostability. The results show that, although CBM mannosylation does not induce major conformational changes, it can increase the thermolysin cleavage resistance up to 50-fold depending on the number of mannose units on the CBM and the attachment site. O-Mannosylation also increases the thermostability of CBM glycoforms up to 16°C, and a mannose disaccharide at Ser3 seems to have the largest themostabilizing effect. Interestingly, the glycoforms with small glycans at each site displayed higher binding affinities for crystalline cellulose, and the glycoform with a single mannose at each of three positions conferred the highest affinity enhancement of 7.4-fold. Overall, by combining chemical glycoprotein synthesis and functional studies, we show that specific glycosylation events confer multiple beneficial properties on Family 1 CBMs. chemical synthesis | cellulase | biofuels | protein engineering

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Isolated fungal cellulose terminal domains and a synthetic minimum analogue bind to cellulose

Cited by 80 publications

References 23 publications

The difference in affinity between two fungal cellulose‐binding domains is dominated by a single amino acid substitution

The difference in affinity between two fungal cellulose‐binding domains is dominated by a single amino acid substitution

The cellulose-binding domain of the major cellobiohydrolase of Trichoderma reesei exhibits true reversibility and a high exchange rate on crystalline cellulose.

Specificity of O -glycosylation in enhancing the stability and cellulose binding affinity of Family 1 carbohydrate-binding modules

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