Crystal structure and functional characterization of an oligosaccharide dehydrogenase from Pycnoporus cinnabarinus provides insights into fungal breakdown of lignocellulose

Background Fungal glucose dehydrogenases (GDHs) are FAD-dependent enzymes belonging to the glucose-methanol-choline oxidoreductase superfamily. These enzymes are classified in the “Auxiliary Activity” family 3 (AA3) of the Carbohydrate-Active enZymes database, and more specifically in subfamily AA3_2, that also includes the closely related flavoenzymes aryl-alcohol oxidase and glucose 1-oxidase. Based on sequence similarity to known fungal GDHs, an AA3_2 enzyme active on glucose was identified in the genome of Pycnoporus cinnabarinus, a model Basidiomycete able to completely degrade lignin. Results In our work, substrate screening and functional characterization showed an unexpected preferential activity of this enzyme toward oligosaccharides containing a β(1→3) glycosidic bond, with the highest efficiency observed for the disaccharide laminaribiose. Despite its sequence similarity to GDHs, we defined a novel enzymatic activity, namely oligosaccharide dehydrogenase (ODH), for this enzyme. The crystallographic structures of ODH in the sugar-free form and in complex with glucose and laminaribiose unveiled a peculiar saccharide recognition mechanism which is not shared with previously characterized AA3 oxidoreductases and accounts for ODH preferential activity toward oligosaccharides. The sugar molecules in the active site of ODH are mainly stabilized through CH-π interactions with aromatic residues rather than through hydrogen bonds with highly conserved residues, as observed instead for the fungal glucose dehydrogenases and oxidases characterized to date. Finally, three sugar-binding sites were identified on ODH external surface, which were not previously observed and might be of importance in the physiological scenario. Conclusions Structure–function analysis of ODH is consistent with its role as an auxiliary enzyme in lignocellulose degradation and unveils yet another enzymatic function within the AA3 family of the Carbohydrate-Active enZymes database. Our findings allow deciphering the molecular determinants of substrate binding and provide insight into the physiological role of ODH, opening new perspectives to exploit biodiversity for lignocellulose transformation into fuels and chemicals.

Identification of an Oligosaccharide Dehydrogenase from Pycnoporus Cinnabarinus Provides Insights into Fungal Breakdown of Lignocellulose

Background: Fungal glucose dehydrogenases (GDHs) are FAD-dependent enzymes belonging to the glucose-methanol-choline oxidoreductase superfamily. These enzymes are classified in the “Auxiliary Activity” family 3 (AA3) of the Carbohydrate-Active enZymes database, and more specifically in subfamily AA3_2, that also includes the closely related flavoenzymes aryl-alcohol oxidase and glucose 1-oxidase. Based on sequence similarity to known fungal GDHs, an AA3_2 enzyme active on glucose was identified in the genome of Pycnoporus cinnabarinus, a model Basidiomycete able to completely degrade lignin.Results: In our work, substrate screening and functional characterization showed an unexpected preferential activity of this enzyme toward oligosaccharides containing a b(1à3) glycosidic bond, with the highest efficiency observed for the disaccharide laminaribiose. Despite its sequence similarity to GDHs, we defined a novel enzymatic activity, namely oligosaccharide dehydrogenase (ODH), for this enzyme. The crystallographic structures of ODH in the sugar-free form and in complex with glucose and laminaribiose unveiled a peculiar saccharide recognition mechanism which is not shared with previously characterized AA3 oxidoreductases and accounts for ODH preferential activity toward oligosaccharides. The sugar molecules in the active site of ODH are mainly stabilized through CH-p interactions with aromatic residues rather than through hydrogen bonds with highly conserved residues, as observed instead for the fungal glucose dehydrogenases and oxidases characterized to date. Finally, three sugar-binding sites were identified on ODH external surface, which were not previously observed and might be of importance in the physiological scenario.Conclusions: Structure-function analysis of ODH is consistent with its role as an auxiliary enzyme in lignocellulose degradation and unveils yet another enzymatic function within the AA3 family of the Carbohydrate-Active enZymes database. Our findings allow deciphering the molecular determinants of substrate binding and provide insight into the physiological role of ODH, opening new perspectives to exploit biodiversity for lignocellulose transformation into fuels and chemicals.

Foot Print of Macro Fungi in The Coastal Forest of Bama, Baluran National Park, East Java

Baluran National Park, West Java, as one of the conservation sites in Indonesia, has the attraction of the varied types of ecosystems, including fungi. This study aimed to analyze the diversity of fungi in Bama Coastal Forest, Baluran National Park. The method was explorative with plot purposive sampling technique. Parameters in this study include abundance, dominance, and diversity of fungi enriched with physical parameters of humidity and temperature. The fungi were documented and macroscopically observed. Data were analyzed using the abundance index, dominance index, and diversity index. This research identified 18 types of macrofungi in Bama Coastal forest, Baluran National Park East Java including Ganoderma, sp, Hexagonia tenuis, Trametes hirsute, Phellinus sp.1 and sp.2, Ganoderma applanatum, Phellinus igniarius, Pycnoporus cinnabarinus, Daedalea quercina, Tyromyces chioneus, Microporus xanthopus, Calvatia sp., Irpex lacteus, Trichaptum sp., Lentinus sp. Poria corticola, Tyromyces sp., and Lichemomphalia sp. One fungi species (Ganoderma sp.) has the highest abundance index (27.62). The Medium abundance index was found in three species (Hexagonia tenuis, Tyromyces chioneus, Tyromyces sp.) while the rest fungi were in low abundance index. Dominance index of all fungi species identified in Bama was in low category (D<0.5) with medium diversity (2.30). It can be concluded that the diversity of microfungi in the coastal forest of Bama, Baluran National park was a distribution medium of individual of each species and medium community stability. Environmental conditions in the Bama Coast Forest with 93% humidity and an average air temperature of 26-27oC could support for the fungi to grow quite well.

EKSPLORASI JAMUR MAKROSKOPIS DI HUTAN OELUAN KABUPATEN TIMOR TENGAH UTARA

Penelitian mengenai keanekaragaman jamur makroskopis di kawasan Hutan Oeluan kabupaten Timor tengah Utara Belum pernah dilaporkan. Penelitian ini telah dilaksanakan pada bulan Januari 2020 dengan letak geografis 9° 2¢ 48" LS- 9° 37¢ 36" LS124° 4¢ 2" BT- 124°46¢ 0" BT, penelitian ini dilakukan pada empat jalur yang berbeda. Panjang jalur pengamatan adalah 100 m. Metode yang dilakuan pada setiap Jalur pemgamatan di setiap adalah metode jelajah dan Purpossive sampling, berdasarkan keberadaan jamur makroskopis yang dianggap mewakili kawasan tersebut, setelah itu dilanjutkan dengan mencatat jumlah individu, koleksi dan dokumentasi. Pengamatan dan pengkoleksian spesies dilakukan secara eksploratif. Dari hasil penelitian ditemukan 9 spesies jamur makroskopis yaitu, Ganoderma aplatum, Earliella scabrosa, Polyporus tenuiculus, Polyporus squamosus, Mikroporus xanthopus, Favolus grammocephalus, Pycnoporus cinnabarinus, Ganoderma lucidium, Laccaria vinaceoavellaneae

New mycological knowledge on territory of NNP “Hutsulshchyna”

The article presents the results of mycological research during 2018 on the territory of the National Nature Park “Hutsulshchyna”. The main goal was to replenish the macromycetes lists and search for new localities of rare fungi. Field research were conducted out on the seized area of the NNP “Hutsulshchyna” by route-expeditionary method. As a result, 323 species of mushrooms and mushroom organisms were identified. A systematic analysis of the species and their distribution in ecological and trophic groups has been made. Data on 32 new species of mycobiota for NNP “Hutsulshchyna” are presented: Leotia lubrica (Scop.) Pers., Rutstroemia conformata (P. Karst.) Nannf., Amanita mairei Foley, Cortinarius torvus (Fr.) Fr., Phaeoclavulina abietina (Pers.) Giachini, Aphroditeola olida (Quél.) Redhead & Manfr. Binder, Inocybe adaequata (Britzelm.) Sacc., Ossicaulis lignatilis (Pers.) Redhead & Ginns, Gymnopus foetidus (Sowerby) P.M. Kirk , Pterula multifida (Chevall.) Fr., Leratiomyces squamosus (Pers.) Bridge & Spooner, Clitocybe costata Kühner & Romagn., Clitocybe pruinosa P. Kumm., Lepista luscina (Fr.) Singer, Melanoleuca melaleuca (Pers.) Murrill, Phyllotopsis nidulans (Pers.) Singer, Tricholoma equestre (L.) P. Kumm., Leccinum piceinum Pilát & Dermek, Rhizopogon roseolus (Corda) Th. Fr., Cantharellus ferruginascens P.D. Orton, Craterellus foetidus A.H. Sm., Coltricia cinnamomea (Jacq.) Murrill, Byssomerulius corium (Pers.) Parmasto, Climacodon septentrionalis (Fr.) P. Karst., Picipes melanopus (Pers.) Zmitr. & Kovalenko, Pycnoporus cinnabarinus (Jacq.) P. Karst., Sarcodontia pachyodon (Pers.) Spirin, Tyromyces chioneus (Fr.) P. Karst., Lactarius fluens Boud., Russula graveolens Romell, Russula violacea Quél. and rare species Leucoagaricus nympharum (Kalchbr.) Bon, which was first discovered in the park. Details of the location of all new macromycetes, their ecology and distribution. To date, 1031 species of macromycetes are known for the NNP “Hutsulshchyna”, among them 18 species are rare and included in the Red Data Book of Ukraine.

Acción antifúngica de clorpromacina, ketoconazol y cicloheximida sobre dos cepas de pycnoporus cinnabarinus (Jacq. Ex fr.) Karst.

El desarrollo de dos cepas de Pycnoporus cinnabarinus (Jacq. a Fr.) Kant, fue inhibido durante 72 horas al agregar al medio de cultivo clorpromacina, ketoconazol o cicloherimida respectivamente. La cepa Pc58 fue inhibida por 33 ug/ml de clorpromacina y la cepa Pc470.6 por 40 ug/ml. Cicloherimida inhibió el crecimiento de la cepa Pc58 a una concentración de 3 ug/ml y a la cepa Pc470.6 a 5 ug/ml. El agente más eficaz para inhibir el crecimiento de estas cepas de P. cinnabarinus fue el ketoconazol. que actuó a concentraciones menores de 0.5 ug/ml en la cepa Pc58 y a 0.9 ug/ml en la cepa Pc470.6.

Compatibilidad sexual entre pycnoporus cinnabarinus var. Osorninus y pycnoporus sanguineus

Se estudia la compatibilidad sexual entre cepas chilenas de Pycnoporus cinnabarinus var. Osorninus, con una cepa P. cinnabarinus var. cinnabarinus obtenida del laboratorio del Dr. Antonio Martínez del Centro de Biología Celular, Madrid, España, y cepas de P. sanguineus de Argentina y Uruguay, a partir de fructificaciones producidas en el laboralorio y cruzando los micelios monospóricos entre sí para obtener los polos de coda cepa. Las cepas chilenas (P. cinnabarinus var. Osorninus) son heterotálicas tetrapolares, incompatibles con la cepa española de P. cinnabarinus var. cinnabarinus, pero si compatibles con las cepas de P. sanguineus (Argentina y Uruguay), dando fructificaciones fértiles. Los resultados obtenidos determinan que P. cinnabarinus var. Osorninus debe ser considerada como P. sanguineus.

Nueva variedad chilena de pycnoporus cinnabarinus (jacq. Ex.fr.) Karst. Estudio de sus características morfológicas y fisiológicas.

Se describe la variedad ligninolítica Pycnoporus cinnabarinus (Jacq.ex Fr.) Kant. var. osorninus Burgos var. nov. basándonos en que sus características morfológicas difieren claramente de P. cinnabarinus. Además esta variedad tiene una tasa de crecimiento superior a la de P. cinnabarinus. El medio liquido con sulfato de amonio como fuente de nitrógeno estimula la formación de clamidosporas fructificacines en agar-extracto de malta o medios con aserrín de pino forman tubos con 4 poros/mm.