The Saccharomyces cerevisiae quinone oxidoreductase Lot6p: stability, inhibition and cooperativity.

Lot6p (EC 1.5.1.39; Ylr011wp) is the sole quinone oxidoreductase in the budding yeast, Saccharomyces cerevisiae. Using hexahistidine tagged, recombinant Lot6p, we determined the steady-state enzyme kinetic parameters with both NADH and NADPH as electron donors; no cooperativity was observed with these substrates. The NQO1 inhibitor curcumin, the NQO2 inhibitor resveratrol, the bacterial nitroreductase inhibitor nicotinamide and the phosphate mimic vanadate all stabilise the enzyme towards thermal denaturation as judged by differential scanning fluorimetry. All except vanadate have no observable effect on the chemical cross-linking of the two subunits of the Lot6p dimer. These compounds all inhibit Lot6p's oxidoreductase activity, and all except nicotinamide exhibit negative cooperativity. Molecular modelling suggests that curcumin, resveratrol and nicotinamide all bind over the isoalloxazine ring of the FMN cofactor in Lot6p. Resveratrol was predicted to contact an α-helix that links the two active sites. Mutation of Gly-142 (which forms part of this helix) to serine does not greatly affect the thermal stability of the enzyme. However, this variant shows less cooperativity towards resveratrol than the wild type. This suggests a plausible hypothesis for the transmission of information between the subunits and, thus, the molecular mechanism of negative cooperativity in Lot6p.

Genomic insight into pathogenicity of dematiaceous fungus Corynespora cassiicola.

Corynespora cassiicola is a common plant pathogen that causes leaf spot disease in a broad range of crop, and it heavily affect rubber trees in Malaysia (Hsueh, 2011; Nghia et al., 2008). The isolation of UM 591 from a patient's contact lens indicates the pathogenic potential of this dematiaceous fungus in human. However, the underlying factors that contribute to the opportunistic cross-infection have not been fully studied. We employed genome sequencing and gene homology annotations in attempt to identify these factors in UM 591 using data obtained from publicly available bioinformatics databases. The assembly size of UM 591 genome is 41.8 Mbp, and a total of 13,531 (≥99 bp) genes have been predicted. UM 591 is enriched with genes that encode for glycoside hydrolases, carbohydrate esterases, auxiliary activity enzymes and cell wall degrading enzymes. Virulent genes comprising of CAZymes, peptidases, and hypervirulence-associated cutinases were found to be present in the fungal genome. Comparative analysis result shows that UM 591 possesses higher number of carbohydrate esterases family 10 (CE10) CAZymes compared to other species of fungi in this study, and these enzymes hydrolyses wide range of carbohydrate and non-carbohydrate substrates. Putative melanin, siderophore, ent-kaurene, and lycopene biosynthesis gene clusters are predicted, and these gene clusters denote that UM 591 are capable of protecting itself from the UV and chemical stresses, allowing it to adapt to different environment. Putative sterigmatocystin, HC-toxin, cercosporin, and gliotoxin biosynthesis gene cluster are predicted. This finding have highlighted the necrotrophic and invasive nature of UM 591.

Identification and characterization of Daldinia eschscholtzii isolated from skin scrapings, nails, and blood.

BACKGROUND: Daldinia eschscholtzii is a filamentous wood-inhabiting endophyte commonly found in woody plants. Here, we report the identification and characterization of nine D. eschscholtzii isolates from skin scrapings, nail clippings, and blood. METHODS: The nine isolates were identified based on colony morphology, light microscopy, and internal transcribed spacer (ITS)-based phylogeny. In vitro antifungal susceptibility of the fungal isolates was evaluated by the Etest to determine the minimum inhibitory concentration (MIC). RESULTS: The nine isolates examined were confirmed as D. eschscholtzii. They exhibited typical features of Daldinia sp. on Sabouraud Dextrose Agar, with white felty colonies and black-gray coloration on the reverse side. Septate hyphae, branching conidiophore with conidiogenous cells budding from its terminus, and nodulisporium-like conidiophores were observed under the microscope. Phylogenetic analysis revealed that the nine isolates were clustered within the D. eschscholtzii species complex. All the isolates exhibited low MICs against azole agents (voriconazole, posaconazole, itraconazole, and ketoconazole), as well as amphotericin B, with MIC of less than 1 µg/ml. DISCUSSION: Early and definitive identification of D. eschscholtzii is vital to reducing misuse of antimicrobial agents. Detailed morphological and molecular characterization as well as antifungal profiling of D. eschscholtzii provide the basis for future studies on its biology, pathogenicity, and medicinal potential.