Structure, computational and biochemical analysis of PcCel45A endoglucanase from Phanerochaete chrysosporium and catalytic mechanisms of GH45 subfamily C members

Godoy, Andre S.; Pereira, Caroline S.; Ramia, Marina Paglione; Silveira, Rodrigo L.; Camilo, César M.; Kadowaki, Marco Antonio Seiki; Lange, Lene; Busk, Peter Kamp; Nascimento, Alessandro S.; Skaf, Munir S.; Polikarpov, Igor

doi:10.1038/s41598-018-21798-9

Cited by 16 publications

(16 citation statements)

References 48 publications

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“…This is an example of the ability of CUPP to capture differences in substrate specificities within this subfamily. Similarly, it was reported that peptide-based clustering of GH45 (by PPR) divided the protein into groups and captured differences in their 3D structure [32]. Biochemical activity testing also supported the distinction of these groups.…”

Section: Discussionmentioning

confidence: 62%

Peptide-based functional annotation of carbohydrate-active enzymes by conserved unique peptide patterns (CUPP)

Barrett

Lange²

2019

Biotechnol Biofuels

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Background Insight into the function of carbohydrate-active enzymes is required to understand their biological role and industrial potential. There is a need for better use of the ample genomic data in order to enable selection of the most interesting proteins for further studies. The basis for elaborating a new approach to sequence analysis is the hypothesis that when using conserved peptide patterns to determine the similarities between proteins, the exact spacing between conserved adjacent amino acids in the proteins plays a prominent functional role. Thus, the objective of developing the method of conserved unique peptide patterns (CUPP) is to construct a peptide-based grouping and validate the method to provide evidence that CUPP captures function-related features of the individual carbohydrate-active enzymes (as defined by CAZy families). This approach facilitates grouping of enzymes at a level lower than protein families and/or subfamilies. A standardized, efficient, and robust approach to functional annotation of carbohydrate-active enzymes would support improved molecular insight into enzyme–substrate interaction. Results A new nonalignment-based clustering and functional annotation tool was developed that uses conserved unique peptides patterns to perform automated clustering of proteins and formation of protein groups. A peptide-based model was constructed for each of these protein CUPP groups to be used to automatically annotate protein family, subfamily, and EC function of carbohydrate-active enzymes. CUPP prediction can annotate proteins (from any CAZy family) with high F-score to existing family (0.966), subfamily (0.961), and EC-function (0.843). The speed of the CUPP program was estimated and exemplified by prediction of the 504,017 nonredundant proteins of CAZy in less than four CPU hours. Conclusion It was possible to construct an automated system for clustering proteins within families and use the resulting CUPP groups to directly build peptide-based models for genome annotation. The CUPP runtime, F-score, sensitivity, and precisions of family and subfamily annotations match or represent an improvement compared to state-of-the-art tools. The speed of the CUPP annotation is similar to the rapid DIAMOND annotation tool. CUPP facilitates automated annotation of full genome assemblies to any CAZy family. Electronic supplementary material The online version of this article (10.1186/s13068-019-1436-5) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 62%

Peptide-based functional annotation of carbohydrate-active enzymes by conserved unique peptide patterns (CUPP)

Barrett

Lange²

2019

Biotechnol Biofuels

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“…Indicating activity on β-1,4 linkages is the primary mechanism of action. According to previously reports, expansins have higher similarity with GH45 subfamily C enzymes than with other members of the GH45 family [31,32]. Studies indicating expansins with higher similarity with GH45 subfamily C enzymes than others have a motif termed HFD as part of its active site, which is also present in TlSWO (HMD).…”

Section: Discussionmentioning

confidence: 95%

A novel Swollenin from Talaromyces leycettanus JCM12802 enhances cellulase hydrolysis towards various substrates

Zhang

Wang

Brunecky

et al. 2020

Preprint

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Background: Swollenins exist within some fungal species such as Trichoderma reesei and Aspergillus niger, and are candidate accessory enzymes for the biodegradation of cellulosic substrates. Swollenins theorized role in biological processes is as promoters in the rearrangement of non-covalent interaction networks formed by cell wall polysaccharides. Such network rearrangements though to be responsible for expanding accessibility for hydrolytic enzyme degradation processes. We hereby report in detail the characterization of a recombinant swollenin and its disruptive activity on cellulosic substrates and its synergistic effects while interacting with cellulases.Results: We have expressed a novel swollenin gene Tlswo consists of an open reading frame encoding 503 amino acids to be identified from Talaromyces leycettanus JCM12802 and successfully expressed it in both Trichoderma reesei and Pichia pastoris. Similar to other fungal swollenins, TlSWO is composed of a N-terminal family 1 carbohydrate binding module (CBM1) followed by a Ser/Thr rich linker connected to an expansin-like domain, followed by a family of 45 endoglucanase-like domains and a group-2 grass pollen allergen domain. From our experimental results, TlSWO demonstrated disruptive activity on Avicel and displayed a synergistic effect with cellobiohydrolases, enhancing its hydrolytic performance up to 132%. We also explored the activity of TlSWO on several model substrates as well as pretreated biomass. Our results indicate that TlSWO is capable of releasing reducing sugars from lichenan, barley β-glucan, carboxymethyl cellulose sodium (CMC-Na) and laminarin. The specific activity of TlSWO towards the substates mentioned above is 9.0 ± 0.100 U/mg, 8.9 ± 0.100U/mg, 2.3 ± 0.002 U/mg and 0.79 ± 0.002 U/mg, respectively. Moreover, further experimentation confirms the fungal swollenin TlSWO exhibits maximum activity at pH 4.0 and 50 ℃.Conclusion: This study reports on a novel swollenin protein capable of enhancing biomass conversion. Experimental data reveals the functional diversity of this swollenin with considerable activity on various substrates. Although the exact catalytic mechanism of swollenin’s remains unknown, the functional diversity of TlSWO broadens its applicability under experimental settings, and suggests it may be a promising candidate for future industrial applications.

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“…This indicates that the primary mechanism of action of TlSWO is via activity on β-1,4 linkages. Expansins are more highly similar to GH45 subfamily C enzymes than to other members of the GH45 family ( Igarashi et al, 2008 ; Godoy et al, 2018 ). These expansins have a HFD motif termed as part of their active site, and this motif is also present in Tl SWO (HMD).…”

Section: Discussionmentioning

confidence: 99%

A Swollenin From Talaromyces leycettanus JCM12802 Enhances Cellulase Hydrolysis Toward Various Substrates

et al. 2021

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Swollenins exist within some fungal species and are candidate accessory proteins for the biodegradation of cellulosic substrates. Here, we describe the identification of a swollenin gene, Tlswo, in Talaromyces leycettanus JCM12802. Tlswo was successfully expressed in both Trichoderma reesei and Pichia pastoris. Assay results indicate that TlSWO is capable of releasing reducing sugars from lichenan, barley β-glucan, carboxymethyl cellulose sodium (CMC-Na) and laminarin. The specific activity of TlSWO toward lichenan, barley β-glucan, carboxymethyl cellulose sodium (CMC-Na) and laminarin is 9.0 ± 0.100, 8.9 ± 0.100, 2.3 ± 0.002 and 0.79 ± 0.002 U/mg, respectively. Additionally, TlSWO had disruptive activity on Avicel and a synergistic effect with cellobiohydrolases, increasing the activity on pretreated corn stover by up to 72.2%. The functional diversity of TlSWO broadens its applicability in experimental settings, and indicating that it may be a promising candidate for future industrial applications.

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Structure, computational and biochemical analysis of PcCel45A endoglucanase from Phanerochaete chrysosporium and catalytic mechanisms of GH45 subfamily C members

Cited by 16 publications

References 48 publications

Peptide-based functional annotation of carbohydrate-active enzymes by conserved unique peptide patterns (CUPP)

Peptide-based functional annotation of carbohydrate-active enzymes by conserved unique peptide patterns (CUPP)

A novel Swollenin from Talaromyces leycettanus JCM12802 enhances cellulase hydrolysis towards various substrates

A Swollenin From Talaromyces leycettanus JCM12802 Enhances Cellulase Hydrolysis Toward Various Substrates

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