Characterization of Fungal FAD-Dependent AA3_2 Glucose Oxidoreductases from Hitherto Unexplored Phylogenetic Clades

Wijayanti, Sudarma Dita; Sützl, Leander; Duval, Adèle; Haltrich, Dietmar

doi:10.3390/jof7100873

Cited by 10 publications

(33 citation statements)

References 34 publications

(58 reference statements)

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“…This analysis revealed that β- d -Glc p -(1,6)- d -Glc (G6G, also called gentiobiose), followed by β- d -Glc p -(1,3)- d -Glc (G3G; laminaribiose) and G3G3G (laminaritriose) were the preferred substrates. These results are in good agreement with those reported by Cerutti et al (27) and Wijayanti et al (31). As in those two works, no activity on either cellobiose (G4G), cellotriose (G4G4G) or the trisaccharide G3G4G could be measured, whereas some activity was retained on the mixed trisaccharide G4G3G containing a β-(1,3) glycosidic bond between the reducing end glucose unit and the adjacent unit ( Fig.…”

Section: Resultssupporting

confidence: 94%

“…5B ). No oxidase activity could be detected, confirming thereby the strict dehydrogenase nature of the enzyme as previously observed in hereinbefore mentioned studies (26, 27, 31).…”

Section: Resultssupporting

confidence: 89%

“…Together with previously published work (27, 31), we show that both Pc ODH and Um AA3_2-A appear to form an evolutionarily distinct clade between AA3_2 GOx and GDH, associated to a new substrate specificity. Enzyme kinetics tell us that G6G and G3G are > one order of magnitude better substrates than glucose.…”

Section: Discussionsupporting

confidence: 87%

“…5B). No oxidase activity could be detected, confirming thereby the strict dehydrogenase nature of the enzyme as previously observed in hereinbefore mentioned studies (26,27,31). We then determined the dehydrogenase kinetic parameters of UmAA3_2-A, at optimum pH, for glucose, G3G and G6G (Fig.…”

Section: Umaa3_2-a Is a Dehydrogenase Active On β-13 And β-16-glucool...supporting

confidence: 85%

“…S9A ). During the preparation of this manuscript, Wijayanti et al reported the production and preliminary characterization of several AA3_2s, including Um AA3_2-A (called there Um GDHIII; (31)), for which they observed the same atypical, cleavage and polymerization pattern under SDS-PAGE denaturating conditions. We carried out size exclusion chromatography and showed a unique, monodisperse peak, corresponding to an estimated monomeric size of 48 kDa ( Fig.…”

Section: Resultsmentioning

confidence: 99%

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The maize pathogen Ustilago maydis secretes glycoside hydrolases and carbohydrate oxidases directed towards components of the fungal cell wall

Bissaro

Berrin

Grisel

et al. 2022

Preprint

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Filamentous fungi are keystone microorganisms in the regulation of many processes occurring on Earth, such as plant biomass decay, pathogenesis as well as symbiotic associations. In many of these processes, fungi secrete carbohydrate-active enzymes (CAZymes) to modify and/or degrade carbohydrates. Ten years ago, while evaluating the potential of a secretome from the maize pathogen Ustilago maydis to supplement lignocellulolytic cocktails, we noticed it contained many unknown or poorly characterized CAZymes. Here, and after re-annotation of this dataset and detailed phylogenetic analyses, we observed that several CAZymes (including glycoside hydrolases and carbohydrate oxidases) are predicted to act on the fungal cell wall (FCW), notably on β-1,3-glucans. We heterologously produced and biochemically characterized two new CAZymes, called UmGH16_1-A and UmAA3_2-A. We show that UmGH16_1-A displays β-1,3-glucanase activity, with a preference for β-1,3-glucans with short β-1,6 substitutions, and UmAA3_2-A is a dehydrogenase catalyzing the oxidation of β-1,3- and β-1,6-gluco-oligosaccharides into the corresponding aldonic acids. Working on model β-1,3-glucans, we show that the linear oligosaccharide products released by UmGH16_1-A are further oxidized by UmAA3_2-A, bringing to light a putative biocatalytic cascade. Interestingly, analysis of available transcriptomics data indicates that both UmGH16_1-A and UmAA3_2-A are co-expressed, only during early stages of U. maydis infection cycle. Altogether, our results suggest that both enzymes are connected and that additional accessory activities still need to be uncovered to fully understand the biocatalytic cascade at play and its physiological role.ImportanceFilamentous fungi play a central regulatory role on Earth, notably in the global carbon cycle. Regardless of their lifestyle, filamentous fungi need to remodel their own cell wall (mostly composed of polysaccharides) to grow and proliferate. To do so, they must secrete a large arsenal of enzymes, most notably carbohydrate-active enzymes (CAZymes). However, research on fungal CAZymes over past decades has mainly focused on finding efficient plant biomass conversion processes while CAZymes directed at the fungus itself have remained little explored. In the present study, using the maize pathogen Ustilago maydis as model, we set off to evaluate the prevalence of CAZymes directed towards the fungal cell wall during growth of the fungus on plant biomass and characterized two new CAZymes active on fungal cell wall components. Our results suggest the existence of a biocatalytic cascade that remains to be fully understood.

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Section: Resultssupporting

confidence: 94%

“…5B ). No oxidase activity could be detected, confirming thereby the strict dehydrogenase nature of the enzyme as previously observed in hereinbefore mentioned studies (26, 27, 31).…”

Section: Resultssupporting

confidence: 89%