Biochemical Characterization and Mutational Analysis of a Lactone Hydrolase from Phialophora americana.

The mycotoxin zearalenone (ZEN) is a secondary metabolite produced mainly by Fusarium species. ZEN poses health hazards both for humans and animals, as a major contaminant in the food and feed industries. Currently, there is no effective technique for degrading ZEN during industrial processes. In this study, we isolated and biochemically characterized a novel lactone hydrolase, ZHD607, isolated from Phialophora americana, cloned, and exogenously expressed in Pichia pastoris. ZHD607 was characterized as a mesophilic lactone hydrolase having a neutral pH and showing optimal activity at 35 °C and pH 8.0. Two mutants, ZHDM1 and I160Y, generated from ZHD607 based on structure and sequence alignment analyses, exhibited 2.9- and 3.4-fold higher activity towards ZEN than did ZHD607. Molecular dynamics simulation revealed diverse mechanisms driving this improved catalytic activity. These findings enrich our knowledge about ZHD enzyme family and represent an important step toward industrialization of ZEN-detoxifying lactone hydrolases.

Melanin: Quantification and protection against oxidative stress in chromoblastomycosis agents.

Chromoblastomycosis (CBM) is a chronic cutaneous and subcutaneous infection caused by melanized fungal species. We quantified the extractable melanin of 77 strains of CBM agents distributed within five genera. Moreover, resistance to oxidative stress was evaluated in strains exposed or not to the melanin inhibitor tricyclazole. The median percentage of melanin mass extracted from dry fungal mass varied from 0.69 (Rhinocladiella similis) to 3.81 (Phialophora americana). Inhibition of melanin synthesis decreased survival rates to hydrogen peroxide. Together, these data highlight the importance of melanin in CBM agents.

Cryo-EM structures of fungal and metazoan mitochondrial calcium uniporters.

The mitochondrial calcium uniporter (MCU) is a highly selective calcium channel and a major route of calcium entry into mitochondria. How the channel catalyses ion permeation and achieves ion selectivity are not well understood, partly because MCU is thought to have a distinct architecture in comparison to other cellular channels. Here we report cryo-electron microscopy reconstructions of MCU channels from zebrafish and Cyphellophora europaea at 8.5 Å and 3.2 Å resolutions, respectively. In contrast to a previous report of pentameric stoichiometry for MCU, both channels are tetramers. The atomic model of C. europaea MCU shows that a conserved WDXXEP signature sequence forms the selectivity filter, in which calcium ions are arranged in single file. Coiled-coil legs connect the pore to N-terminal domains in the mitochondrial matrix. In C. europaea MCU, the N-terminal domains assemble as a dimer of dimers; in zebrafish MCU, they form an asymmetric crescent. The structures define principles that underlie ion permeation and calcium selectivity in this unusual channel.

A Pair of New Polyketide Enantiomers from Three Endolichenic Fungal Strains Nigrospora sphaerica, Alternaria alternata, and Phialophora sp.

A pair of new enantiomeric polyketides, (-)- and (+)-nigrosporaol A (1a and 1b), along with one related known compound, (+)-alternarienoic acid (2), were isolated from three endolichenic fungal strains, Nigrospora sphaerica (No.83-1-1-2), Alternaria alternata (No.58-8-4-1), and Phialophora sp.(No.96-1-8-1). Their structures, including the absolute configurations, were elucidated on the basis of spectroscopic methods, X-ray diffraction analysis, and the modified Mosher's method.

Heptaketides with antiviral activity from three endolichenic fungal strains Nigrospora sp., Alternaria sp. and Phialophora sp.

Two new heptaketides, (+)-(2S,3S,4aS)-altenuene (1a) and (-)-(2S,3S,4aR)-isoaltenuene (2a), together with six known compounds, (-)-(2R,3R,4aR)-altenuene (1b), (+)-(2R,3R,4aS)-isoaltenuene (2b), 5'-methoxy-6-methyl-biphenyl-3,4,3'-triol (3), alternariol (4), alternariol-9-methyl ether (5), and 4-hydroxyalternariol-9-methyl ether (6) were isolated from the EtOAc extract of an endolichenic fungal strain Nigrospora sphaerica (No.83-1-1-2). Compounds 1a and 1b were separated from enantiomers 1 by chiral HPLC, and so were 2a and 2b from enantiomers 2. Interestingly, 1-6 were also obtained from other two endolichenic fungal strains Alternaria alternata (No.58-8-4-1) and Phialophora sp. (No.96-1-8-1). The structures of 1-6 were elucidated by means of MS, HR-MS, NMR, and X-ray diffraction. Furthermore, the absolute configurations of 1a-2b were determined by CD experiments and CD calculation. Of these compounds, 4 and 5 showed antiviral activity against herpes simplex virus (HSV) in vitro, with IC(50) values of 13.5 and 21.3 µM, and with selective index (SI) values of 26.5 and 17.1, respectively.

Antineoplastic agents. 488. Isolation and structure of yukonin from a yukon territory fungus.

Cancer cell line bioassay-guided separation of an extract from a Yukon Territory fungus resulted in the isolation of a new C(16)-terpene dilactone designated yukonin (1) accompanied by two previously known, structurally related constituents (2 and 3). The structure of each was determined by X-ray crystallographic analysis. Dilactone 2 was found to correspond to LL-Z1271alpha isolated from fungi in the genera Oidiodendron, Acrostalagmus, and Holwaya, while dilactone 3 had earlier been prepared by reduction of an alpha-lactol derivative. Each of the dilactones was found to inhibit growth of human cancer cell lines (pancreas, breast, CNS, lung, colon, and prostate) and some pathogenic fungi.