A structural study of <i>Hypocrea jecorina</i> Cel5A

Lee, Toni M.; Farrow, Mary F.; Arnold, Frances H.; Mayo, Stephen L.

doi:10.1002/pro.730

Cited by 51 publications

(42 citation statements)

References 29 publications

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“…They hydrolyze ␤-1,4-D-glycosidic bonds randomly and internally of the amorphous region of cellulose. The catalytic core domain of Cel5A from T. reesei determined at 2.05 Å shows a substrate-binding pocket consisting of a deep catalytic cleft within a shallow groove, consistent with other structural studies of GH5 endoglucanases (115). The Thermoascus aurantiacus GH5 endoglucanase, which consists of a catalytic module with compact 8-fold ␤/␣-barrel architecture (116), has a long, tryptophan-rich substrate-binding groove suggesting substrate-binding subsites at positions Ϫ4 to ϩ3, in addition to the two conserved catalytic glutamates (116).…”

Section: Cellulasessupporting

confidence: 88%

Genomics Review of Holocellulose Deconstruction by Aspergilli

Segato

Damásio

Lucas

et al. 2014

Microbiol Mol Biol Rev

113

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SUMMARY Biomass is constructed of dense recalcitrant polymeric materials: proteins, lignin, and holocellulose, a fraction constituting fibrous cellulose wrapped in hemicellulose-pectin. Bacteria and fungi are abundant in soil and forest floors, actively recycling biomass mainly by extracting sugars from holocellulose degradation. Here we review the genome-wide contents of seven Aspergillus species and unravel hundreds of gene models encoding holocellulose-degrading enzymes. Numerous apparent gene duplications followed functional evolution, grouping similar genes into smaller coherent functional families according to specialized structural features, domain organization, biochemical activity, and genus genome distribution. Aspergilli contain about 37 cellulase gene models, clustered in two mechanistic categories: 27 hydrolyze and 10 oxidize glycosidic bonds. Within the oxidative enzymes, we found two cellobiose dehydrogenases that produce oxygen radicals utilized by eight lytic polysaccharide monooxygenases that oxidize glycosidic linkages, breaking crystalline cellulose chains and making them accessible to hydrolytic enzymes. Among the hydrolases, six cellobiohydrolases with a tunnel-like structural fold embrace single crystalline cellulose chains and cooperate at nonreducing or reducing end termini, splitting off cellobiose. Five endoglucanases group into four structural families and interact randomly and internally with cellulose through an open cleft catalytic domain, and finally, seven extracellular β-glucosidases cleave cellobiose and related oligomers into glucose. Aspergilli contain, on average, 30 hemicellulase and 7 accessory gene models, distributed among 9 distinct functional categories: the backbone-attacking enzymes xylanase, mannosidase, arabinase, and xyloglucanase, the short-side-chain-removing enzymes xylan α-1,2-glucuronidase, arabinofuranosidase, and xylosidase, and the accessory enzymes acetyl xylan and feruloyl esterases.

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Section: Cellulasessupporting

confidence: 88%

Genomics Review of Holocellulose Deconstruction by Aspergilli

Segato

Damásio

Lucas

et al. 2014

Microbiol Mol Biol Rev

113

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“…SD indicates the standard deviation from the results from three reactions. (45). Two amino acid residues of C. hutchinsonii Cel5B (E159 and E247) are predicted to be involved in the double-displacement mechanism characteristic of family 5 cellulases (Fig.…”

Section: Figmentioning

confidence: 99%

“…7B). In the H. jecorina Cel5A active site, a catalytic triad acts as a proton shuttle that allows a glutamate residue to protonate the glycosidic bond, promoting its cleavage (45). This catalytic triad is present in C. hutchinsonii Cel5B and consists of E159, the general acid/base catalyst, H219, and T246 (45,46).…”

Section: Figmentioning

confidence: 99%

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Periplasmic Cytophaga hutchinsonii Endoglucanases Are Required for Use of Crystalline Cellulose as the Sole Source of Carbon and Energy

Zhu

Han

Hefferon

et al. 2016

Appl Environ Microbiol

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The soil bacterium Cytophaga hutchinsonii actively digests crystalline cellulose by a poorly understood mechanism. Genome analyses identified nine genes predicted to encode endoglucanases with roles in this process. No predicted cellobiohydrolases, which are usually involved in the utilization of crystalline cellulose, were identified. Chromosomal deletions were performed in eight of the endoglucanase-encoding genes: cel5A, cel5B, cel5C, cel9A, cel9B, cel9C, cel9E, and cel9F. Each mutant retained the ability to digest crystalline cellulose, although the deletion of cel9C caused a modest decrease in cellulose utilization. Strains with multiple deletions were constructed to identify the critical cellulases. Cells of a mutant lacking both cel5B and cel9C were completely deficient in growth on cellulose. Cell fractionation and biochemical analyses indicate that Cel5B and Cel9C are periplasmic nonprocessive endoglucanases. The requirement of periplasmic endoglucanases for cellulose utilization suggests that cellodextrins are transported across the outer membrane during this process. Bioinformatic analyses predict that Cel5A, Cel9A, Cel9B, Cel9D, and Cel9E are secreted across the outer membrane by the type IX secretion system, which has been linked to cellulose utilization. These secreted endoglucanases may perform the initial digestion within amorphous regions on the cellulose fibers, releasing oligomers that are transported into the periplasm for further digestion by Cel5B and Cel9C. The results suggest that both cell surface and periplasmic endoglucanases are required for the growth of C. hutchinsonii on cellulose and that novel cell surface proteins may solubilize and transport cellodextrins across the outer membrane. IMPORTANCEThe bacterium Cytophaga hutchinsonii digests crystalline cellulose by an unknown mechanism. It lacks processive cellobiohydrolases that are often involved in cellulose digestion. Critical cellulolytic enzymes were identified by genetic analyses. Intracellular (periplasmic) nonprocessive endoglucanases performed an important role in cellulose utilization. The results suggest a model involving partial digestion at the cell surface, solubilization and uptake of cellodextrins across the outer membrane by an unknown mechanism, and further digestion within the periplasm. The ability to sequester cellodextrins and digest them intracellularly may limit losses of soluble cellobiose to other organisms. C. hutchinsonii uses an unusual approach to digest cellulose and is a potential source of novel proteins to increase the efficiency of conversion of cellulose into soluble sugars and biofuels. C ytophaga hutchinsonii is a common cellulolytic soil bacterium that belongs to the phylum Bacteroidetes (1). Cells of C. hutchinsonii digest crystalline cellulose and grow with filter paper as the sole source of carbon and energy. C. hutchinsonii specializes in cellulose digestion and is known to grow only on cellulose or cellulose digestion products (1, 2). Direct contact of cells with their insoluble s...

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“…In contrast to synergistic interactions, the reaction mechanism of isolated enzymes is better understood, since structure-function studies have led to the identification of catalytic residues for cellobiohydrolases and endoglucanases (5)(6)(7)(8)(9)(10)(11). Sequence similarity and, therefore, structural properties are at the origin of the classification of these enzymes in the CAZy (Carbohydrate-Active enZymes) database, facilitating the assignment of function (12).…”

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confidence: 99%

Insights into Exo- and Endoglucanase Activities of Family 6 Glycoside Hydrolases from Podospora anserina

Poidevin

Féliu

Doan

et al. 2013

Appl Environ Microbiol

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The ascomycete Podospora anserina is a coprophilous fungus that grows at late stages on droppings of herbivores. Its genome encodes a large diversity of carbohydrate-active enzymes. Among them, four genes encode glycoside hydrolases from family 6 (GH6), the members of which comprise putative endoglucanases and exoglucanases, some of them exerting important functions for biomass degradation in fungi. Therefore, this family was selected for functional analysis. Three of the enzymes, P. anserina Cel6A (PaCel6A), PaCel6B, and PaCel6C, were functionally expressed in the yeast Pichia pastoris. All three GH6 enzymes hydrolyzed crystalline and amorphous cellulose but were inactive on hydroxyethyl cellulose, mannan, galactomannan, xyloglucan, arabinoxylan, arabinan, xylan, and pectin. PaCel6A had a catalytic efficiency on cellotetraose comparable to that of Trichoderma reesei Cel6A (TrCel6A), but PaCel6B and PaCel6C were clearly less efficient. PaCel6A was the enzyme with the highest stability at 45°C, while PaCel6C was the least stable enzyme, losing more than 50% of its activity after incubation at temperatures above 30°C for 24 h. In contrast to TrCel6A, all three studied P. anserina GH6 cellulases were stable over a wide range of pHs and conserved high activity at pH values of up to 9. Each enzyme displayed a distinct substrate and product profile, highlighting different modes of action, with PaCel6A being the enzyme most similar to TrCel6A. PaCel6B was the only enzyme with higher specific activity on carboxymethylcellulose (CMC) than on Avicel and showed lower processivity than the others. Structural modeling predicts an open catalytic cleft, suggesting that PaCel6B is an endoglucanase.

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A structural study of Hypocrea jecorina Cel5A

Cited by 51 publications

References 29 publications

Genomics Review of Holocellulose Deconstruction by Aspergilli

Genomics Review of Holocellulose Deconstruction by Aspergilli

Periplasmic Cytophaga hutchinsonii Endoglucanases Are Required for Use of Crystalline Cellulose as the Sole Source of Carbon and Energy

Insights into Exo- and Endoglucanase Activities of Family 6 Glycoside Hydrolases from Podospora anserina

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