Dynamic light scattering study of the two‐domain structure of <i>Humicola insolens</i> endoglucanase V

Boisset, Claire; Borsali, Rédouane; Schülein, M.; Henrissat, Bernard

doi:10.1016/0014-5793(95)01244-0

Cited by 16 publications

(20 citation statements)

References 24 publications

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“…As a matter of fact the maximum dimensions obtained for CBHI and CBHII from T. reesei were probably overestimated and lead to unrealistic values for the dimension of the linker: ϳ105 Å and ϳ125 Å, respectively for the linkers of CBHI (28 residues) and CBHII (41 residues). By contrast, our results are consistent with the work of Boisset et al (20) on H. insolens Cel45 using dynamic light scattering (estimated D max of the whole protein of 133 Å).…”

Section: Discussionsupporting

confidence: 93%

“…Sample Preparation-H. insolens Cel45 full-length and its isolated catalytic module (Cel45 core) were cloned and expressed in Aspergillus oryzae (24) and purified as already described (20). Site-directed mutagenesis was done using the PCR method.…”

Section: Methodsmentioning

confidence: 99%

“…Finally, a variant having a half-size linker (variant Cel45 ⌬S219-T235) was obtained by deletion of the region coding from Ser-219 to Thr-235. Variants Cel45 ⌬S219-T235 and Cel45 PP, which bear a CBM, were purified using Avicel affinity as described earlier (20). Variant Cel45⌬CBM was purified using ion-exchange chromatography as described for the catalytic module (Cel45 core) (20).…”

Section: Methodsmentioning

confidence: 99%

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Dimension, Shape, and Conformational Flexibility of a Two Domain Fungal Cellulase in Solution Probed by Small Angle X-ray Scattering

Receveur

Czjzek

Schülein

et al. 2002

Journal of Biological Chemistry

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111

102

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Cellulase Cel45 from Humicola insolens has a modular structure with a catalytic module and a cellulose-binding module (CBM) separated by a 36 amino acid, glycosylated, linker peptide. The solution conformation of the entire two domain Cel45 protein as well as the effect of the length and flexibility of the linker on the spatial arrangement of the constitutive modules were studied by small angle x-ray scattering combined with the known three-dimensional structure of the individual modules. The measured dimensions of the enzyme show that the linker exhibits an extended conformation leading to a maximum extension between the two centers of mass of each module corresponding to about four cellobiose units on a cellulose chain. The glycosylation of the linker is the key factor defining its extended conformation, and a five proline stretch mutation on the linker was found to confer a higher rigidity to the enzyme. Our study shows that the respective positioning of the catalytic module and the CBM onto the insoluble substrate is most likely influenced by the linker structure and flexibility. Our results are consistent with a model where cellulases can move on the surface of cellulose with a caterpillar-like displacement with free energy restrictions.

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Section: Discussionsupporting

confidence: 93%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Dimension, Shape, and Conformational Flexibility of a Two Domain Fungal Cellulase in Solution Probed by Small Angle X-ray Scattering

Receveur

Czjzek

Schülein

et al. 2002

Journal of Biological Chemistry

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111

102

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“…The enzyme had two domains (69): a catalytic domain and a cellulose-binding domain joined by a 33-amino-acid linker sequence (40). Endoglucanase 1 (EG1) was a single-domain enzyme composed of two antiparallel ␤-sheets (152).…”

Section: Cellulasementioning

confidence: 99%

Thermophilic Fungi: Their Physiology and Enzymes

Maheshwari

Bharadwaj

Bhat

2000

Microbiol Mol Biol Rev

658

380

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SUMMARY Thermophilic fungi are a small assemblage in mycota that have a minimum temperature of growth at or above 20°C and a maximum temperature of growth extending up to 60 to 62°C. As the only representatives of eukaryotic organisms that can grow at temperatures above 45°C, the thermophilic fungi are valuable experimental systems for investigations of mechanisms that allow growth at moderately high temperature yet limit their growth beyond 60 to 62°C. Although widespread in terrestrial habitats, they have remained underexplored compared to thermophilic species of eubacteria and archaea. However, thermophilic fungi are potential sources of enzymes with scientific and commercial interests. This review, for the first time, compiles information on the physiology and enzymes of thermophilic fungi. Thermophilic fungi can be grown in minimal media with metabolic rates and growth yields comparable to those of mesophilic fungi. Studies of their growth kinetics, respiration, mixed-substrate utilization, nutrient uptake, and protein breakdown rate have provided some basic information not only on thermophilic fungi but also on filamentous fungi in general. Some species have the ability to grow at ambient temperatures if cultures are initiated with germinated spores or mycelial inoculum or if a nutritionally rich medium is used. Thermophilic fungi have a powerful ability to degrade polysaccharide constituents of biomass. The properties of their enzymes show differences not only among species but also among strains of the same species. Their extracellular enzymes display temperature optima for activity that are close to or above the optimum temperature for the growth of organism and, in general, are more heat stable than those of the mesophilic fungi. Some extracellular enzymes from thermophilic fungi are being produced commercially, and a few others have commercial prospects. Genes of thermophilic fungi encoding lipase, protease, xylanase, and cellulase have been cloned and overexpressed in heterologous fungi, and pure crystalline proteins have been obtained for elucidation of the mechanisms of their intrinsic thermostability and catalysis. By contrast, the thermal stability of the few intracellular enzymes that have been purified is comparable to or, in some cases, lower than that of enzymes from the mesophilic fungi. Although rigorous data are lacking, it appears that eukaryotic thermophily involves several mechanisms of stabilization of enzymes or optimization of their activity, with different mechanisms operating for different enzymes.

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“…Endoglucanase V (EGV) and cellobiohydrolase I (CBHI) were purified using Avicel affinity chromatography as described (14). EGV and CBHI gave single bands in SDS-polyacrylamide gel electrophoresis at 43 and 70 kDa, respectively.…”

Section: Enzymesmentioning

confidence: 99%

A Bifunctionalized Fluorogenic Tetrasaccharide as a Substrate to Study Cellulases

Armand

Drouillard

Schülein

et al. 1997

Journal of Biological Chemistry

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Cellulases are usually classified as endoglucanases and cellobiohydrolases, but the heterogeneity of cellulose, in terms of particle size and crystallinity, has always represented a problem for the biochemical characterization of the enzymes. The synthesis of a bifunctionalized tetrasaccharide substrate suitable for measuring cellulase activity by resonance energy transfer is described. The substrate, which carries a 5-(2-aminoethylamino)-1-naphthalenesulfonate group on the non-reducing end and an indolethyl group on the reducing end, was prepared from ␤-lactosyl fluoride and indolethyl ␤-cellobioside by a chemoenzymatic approach using the transglycosylating activity of endoglucanase I of Humicola insolens as the key step. The bifunctionalized substrate has been used for the determination of the catalytic constants of H. insolens endoglucanase I and cellobiohydrolases I and II; this substrate could be of general use to measure the kinetic constants of cellulases able to act on oligomers of degree of polymerization <5. The data also provide evidence that cellobiohydrolases I and II are able to degrade an oligosaccharide substrate carrying non-carbohydrate substituents at both ends.

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Dynamic light scattering study of the two‐domain structure of Humicola insolens endoglucanase V

Cited by 16 publications

References 24 publications

Dimension, Shape, and Conformational Flexibility of a Two Domain Fungal Cellulase in Solution Probed by Small Angle X-ray Scattering

Dimension, Shape, and Conformational Flexibility of a Two Domain Fungal Cellulase in Solution Probed by Small Angle X-ray Scattering

Thermophilic Fungi: Their Physiology and Enzymes

A Bifunctionalized Fluorogenic Tetrasaccharide as a Substrate to Study Cellulases

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