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Substrate specificity and regioselectivity of fungal AA9 lytic polysaccharide monooxygenases secreted by Podospora anserina.

Bennati-Granier C, Garajova S, Champion C, Grisel S, Haon M, Zhou S, Fanuel M, Ropartz D, Rogniaux H, Gimbert I, Record E, Berrin JG - Biotechnol Biofuels (2015)

Bottom Line: Investigation of their regioselective mode of action revealed that PaLPMO9A and PaLPMO9H oxidatively cleaved at both C1 and C4 positions while PaLPMO9E released only C1-oxidized products.Rapid cleavage of cellulose was observed using PaLPMO9H that was the most versatile in terms of substrate specificity as it also displayed activity on cello-oligosaccharides and β-(1,4)-linked hemicellulose polysaccharides (e.g., xyloglucan, glucomannan).This study provides insights into the mode of cleavage and substrate specificities of fungal AA9 LPMOs that will facilitate their application for the development of future biorefineries.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR1163 Biodiversité et Biotechnologie Fongiques, Faculté des Sciences de Luminy, ESIL Polytech, F-13288 Marseille, France ; Polytech Marseille, Aix Marseille Université, F-13288 Marseille, France.

ABSTRACT

Background: The understanding of enzymatic polysaccharide degradation has progressed intensely in the past few years with the identification of a new class of fungal-secreted enzymes, the lytic polysaccharide monooxygenases (LPMOs) that enhance cellulose conversion. In the fungal kingdom, saprotrophic fungi display a high number of genes encoding LPMOs from family AA9 but the functional relevance of this redundancy is not fully understood.

Results: In this study, we investigated a set of AA9 LPMOs identified in the secretomes of the coprophilous ascomycete Podospora anserina, a biomass degrader of recalcitrant substrates. Their activity was assayed on cellulose in synergy with the cellobiose dehydrogenase from the same organism. We showed that the total release of oxidized oligosaccharides from cellulose was higher for PaLPMO9A, PaLPMO9E, and PaLPMO9H that harbored a carbohydrate-binding module from the family CBM1. Investigation of their regioselective mode of action revealed that PaLPMO9A and PaLPMO9H oxidatively cleaved at both C1 and C4 positions while PaLPMO9E released only C1-oxidized products. Rapid cleavage of cellulose was observed using PaLPMO9H that was the most versatile in terms of substrate specificity as it also displayed activity on cello-oligosaccharides and β-(1,4)-linked hemicellulose polysaccharides (e.g., xyloglucan, glucomannan).

Conclusions: This study provides insights into the mode of cleavage and substrate specificities of fungal AA9 LPMOs that will facilitate their application for the development of future biorefineries.

No MeSH data available.


Related in: MedlinePlus

Mass spectrometry analysis of degradation products generated by PaLPMO9H. a Analysis was performed after 16 h of cellulose degradation. The main panel shows the full spectrum of sample with peaks corresponding to native and oxidized cello-oligosaccharides. Fragmented peaks are indicated by arrows. The panels below b, c, and d show the DP4 peaks with m/z value of 687.21, 705.22, and 721.21, respectively, that were fragmented using ESI MS. The oxidized oligosaccharides product species are represented in panels b, c, and d based on the fragmentation patterns. In panel d, the different product species corresponding to the fragmentation pattern are indicated by blue and red dotted bonds
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Fig3: Mass spectrometry analysis of degradation products generated by PaLPMO9H. a Analysis was performed after 16 h of cellulose degradation. The main panel shows the full spectrum of sample with peaks corresponding to native and oxidized cello-oligosaccharides. Fragmented peaks are indicated by arrows. The panels below b, c, and d show the DP4 peaks with m/z value of 687.21, 705.22, and 721.21, respectively, that were fragmented using ESI MS. The oxidized oligosaccharides product species are represented in panels b, c, and d based on the fragmentation patterns. In panel d, the different product species corresponding to the fragmentation pattern are indicated by blue and red dotted bonds

Mentions: As no C4-oxidized standards are available, mass spectrometry was used to confirm the position of oxidation. Analysis of the product mixture generated from PASC with PaLPMO9H confirmed the presence of DP2- to DP5-oxidized and non-oxidized cello-oligosaccharides with products potentially corresponding to a ketone or gem-diol at the non-reducing end (m/z −2 or m/z +16, respectively) and to a lesser extent double-oxidized products (m/z +32) (Fig. 3a). To confirm the position of oxidation generated by PaLPMO9H, electrospray ionisation (ESI) MS/MS was performed on DP4 mono-oxidized product species (ions at m/z +687 and m/z +705, Fig. 3b, c). Interpretation of the fragmentation spectra was based on the previous observation by Isaksen et al. [30] that a gem-diol form at the non-reducing end (C4 position) leads to a double loss of water. This double loss is very clearly observed on the MS2 spectrum of the species at m/z 705.22 (Fig. 3c), suggesting that this species corresponds to a gem-diol form at the non-reducing end. In contrast, a single water loss is observed on Fig. 3b (m/z +687), consistent with a ketone form at the C4 position of the non-reducing end, in addition to several characteristic fragment ions supporting this ketone structure (2,5X3, 1,5X3, 1,5X2). Figure 3d displays the MS2 spectrum recorded for the species at m/z +721.21. Based on the mass accuracy of the instrument, this mass was unequivocally attributed to the sodiated ion of the doubly oxidized DP4. Again, the fragmentation pattern was interpreted following the statements of Isaksen et al. [30], as well as by the observation of some specific fragments. This has led us to propose two structures presumably present as a mixture: in the first one, the two oxidations are brought by the non-reducing end. This is evidenced by the two ions at m/z +527.17 (Y3) and m/z +555.16 (1,5X3), indicating three consecutive non-oxidized glucose units in this structure, thereby suggesting that the fourth one is doubly oxidized. The specific fragment at m/z +555.16 (1,5X3) further indicates that the doubly oxidized unit is the glucose at the non-reducing end. Some fragment ions of the spectrum cannot arise from the previous form and indicate the presence of a second structure. We propose that this structure corresponds to the DP4 in which one oxidation is brought by the non-reducing end while the second one is located at the reducing end. This is supported for example by the ions at m/z +525.15 (B3) and m/z +543.17 (C3), which masses correspond to two consecutive non-modified glucose units and one oxidized glucose unit. Note, however, that the exact positioning of the oxidation at the reducing end could not be ascertained between the C1, C2, C3, or C6 positions. A similar mass spectrometry analysis of the products released from PASC using the PaLPMO9E was performed. It revealed only the presence of C1-oxidized species (Additional file 1: Figure S3).Fig. 3


Substrate specificity and regioselectivity of fungal AA9 lytic polysaccharide monooxygenases secreted by Podospora anserina.

Bennati-Granier C, Garajova S, Champion C, Grisel S, Haon M, Zhou S, Fanuel M, Ropartz D, Rogniaux H, Gimbert I, Record E, Berrin JG - Biotechnol Biofuels (2015)

Mass spectrometry analysis of degradation products generated by PaLPMO9H. a Analysis was performed after 16 h of cellulose degradation. The main panel shows the full spectrum of sample with peaks corresponding to native and oxidized cello-oligosaccharides. Fragmented peaks are indicated by arrows. The panels below b, c, and d show the DP4 peaks with m/z value of 687.21, 705.22, and 721.21, respectively, that were fragmented using ESI MS. The oxidized oligosaccharides product species are represented in panels b, c, and d based on the fragmentation patterns. In panel d, the different product species corresponding to the fragmentation pattern are indicated by blue and red dotted bonds
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4487207&req=5

Fig3: Mass spectrometry analysis of degradation products generated by PaLPMO9H. a Analysis was performed after 16 h of cellulose degradation. The main panel shows the full spectrum of sample with peaks corresponding to native and oxidized cello-oligosaccharides. Fragmented peaks are indicated by arrows. The panels below b, c, and d show the DP4 peaks with m/z value of 687.21, 705.22, and 721.21, respectively, that were fragmented using ESI MS. The oxidized oligosaccharides product species are represented in panels b, c, and d based on the fragmentation patterns. In panel d, the different product species corresponding to the fragmentation pattern are indicated by blue and red dotted bonds
Mentions: As no C4-oxidized standards are available, mass spectrometry was used to confirm the position of oxidation. Analysis of the product mixture generated from PASC with PaLPMO9H confirmed the presence of DP2- to DP5-oxidized and non-oxidized cello-oligosaccharides with products potentially corresponding to a ketone or gem-diol at the non-reducing end (m/z −2 or m/z +16, respectively) and to a lesser extent double-oxidized products (m/z +32) (Fig. 3a). To confirm the position of oxidation generated by PaLPMO9H, electrospray ionisation (ESI) MS/MS was performed on DP4 mono-oxidized product species (ions at m/z +687 and m/z +705, Fig. 3b, c). Interpretation of the fragmentation spectra was based on the previous observation by Isaksen et al. [30] that a gem-diol form at the non-reducing end (C4 position) leads to a double loss of water. This double loss is very clearly observed on the MS2 spectrum of the species at m/z 705.22 (Fig. 3c), suggesting that this species corresponds to a gem-diol form at the non-reducing end. In contrast, a single water loss is observed on Fig. 3b (m/z +687), consistent with a ketone form at the C4 position of the non-reducing end, in addition to several characteristic fragment ions supporting this ketone structure (2,5X3, 1,5X3, 1,5X2). Figure 3d displays the MS2 spectrum recorded for the species at m/z +721.21. Based on the mass accuracy of the instrument, this mass was unequivocally attributed to the sodiated ion of the doubly oxidized DP4. Again, the fragmentation pattern was interpreted following the statements of Isaksen et al. [30], as well as by the observation of some specific fragments. This has led us to propose two structures presumably present as a mixture: in the first one, the two oxidations are brought by the non-reducing end. This is evidenced by the two ions at m/z +527.17 (Y3) and m/z +555.16 (1,5X3), indicating three consecutive non-oxidized glucose units in this structure, thereby suggesting that the fourth one is doubly oxidized. The specific fragment at m/z +555.16 (1,5X3) further indicates that the doubly oxidized unit is the glucose at the non-reducing end. Some fragment ions of the spectrum cannot arise from the previous form and indicate the presence of a second structure. We propose that this structure corresponds to the DP4 in which one oxidation is brought by the non-reducing end while the second one is located at the reducing end. This is supported for example by the ions at m/z +525.15 (B3) and m/z +543.17 (C3), which masses correspond to two consecutive non-modified glucose units and one oxidized glucose unit. Note, however, that the exact positioning of the oxidation at the reducing end could not be ascertained between the C1, C2, C3, or C6 positions. A similar mass spectrometry analysis of the products released from PASC using the PaLPMO9E was performed. It revealed only the presence of C1-oxidized species (Additional file 1: Figure S3).Fig. 3

Bottom Line: Investigation of their regioselective mode of action revealed that PaLPMO9A and PaLPMO9H oxidatively cleaved at both C1 and C4 positions while PaLPMO9E released only C1-oxidized products.Rapid cleavage of cellulose was observed using PaLPMO9H that was the most versatile in terms of substrate specificity as it also displayed activity on cello-oligosaccharides and β-(1,4)-linked hemicellulose polysaccharides (e.g., xyloglucan, glucomannan).This study provides insights into the mode of cleavage and substrate specificities of fungal AA9 LPMOs that will facilitate their application for the development of future biorefineries.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR1163 Biodiversité et Biotechnologie Fongiques, Faculté des Sciences de Luminy, ESIL Polytech, F-13288 Marseille, France ; Polytech Marseille, Aix Marseille Université, F-13288 Marseille, France.

ABSTRACT

Background: The understanding of enzymatic polysaccharide degradation has progressed intensely in the past few years with the identification of a new class of fungal-secreted enzymes, the lytic polysaccharide monooxygenases (LPMOs) that enhance cellulose conversion. In the fungal kingdom, saprotrophic fungi display a high number of genes encoding LPMOs from family AA9 but the functional relevance of this redundancy is not fully understood.

Results: In this study, we investigated a set of AA9 LPMOs identified in the secretomes of the coprophilous ascomycete Podospora anserina, a biomass degrader of recalcitrant substrates. Their activity was assayed on cellulose in synergy with the cellobiose dehydrogenase from the same organism. We showed that the total release of oxidized oligosaccharides from cellulose was higher for PaLPMO9A, PaLPMO9E, and PaLPMO9H that harbored a carbohydrate-binding module from the family CBM1. Investigation of their regioselective mode of action revealed that PaLPMO9A and PaLPMO9H oxidatively cleaved at both C1 and C4 positions while PaLPMO9E released only C1-oxidized products. Rapid cleavage of cellulose was observed using PaLPMO9H that was the most versatile in terms of substrate specificity as it also displayed activity on cello-oligosaccharides and β-(1,4)-linked hemicellulose polysaccharides (e.g., xyloglucan, glucomannan).

Conclusions: This study provides insights into the mode of cleavage and substrate specificities of fungal AA9 LPMOs that will facilitate their application for the development of future biorefineries.

No MeSH data available.


Related in: MedlinePlus