In animals, electron transfer from NADH to molecular oxygen proceeds via large respiratory complexes in a linear respiratory chain. In contrast, most fungi utilise branched respiratory chains. These consist of alternative NADH dehydrogenases, which catalyse rotenone insensitive oxidation of matrix NADH or enable cytoplasmic NADH to be used directly. Many also contain an alternative oxidase that probably accepts electrons directly from ubiquinol. A few fungi lack Complex I. Although the alternative components are non-energy conserving, their organisation within the fungal electron transfer chain ensures that the transfer of electrons from NADH to molecular oxygen is generally coupled to proton translocation through at least one site. The alternative oxidase enables respiration to continue in the presence of inhibitors for ubiquinol:cytochrome c oxidoreductase and cytochrome c oxidase. This may be particularly important for fungal pathogens, since host defence mechanisms often involve nitric oxide, which, whilst being a potent inhibitor of cytochrome c oxidase, has no inhibitory effect on alternative oxidase. Alternative NADH dehydrogenases may avoid the active oxygen production associated with Complex I. The expression and activity regulation of alternative components responds to factors ranging from oxidative stress to the stage of fungal development.
The storage tissues of many plants contain protease inhibitors that are believed to play an important role in defending the plant from invasion by pests and pathogens. These proteinaceous inhibitor molecules belong to a number of structurally distinct families. We describe here the isolation, purification, initial inhibitory properties, and three-dimensional structure of a novel trypsin inhibitor from seeds of Veronica hederifolia (VhTI). The VhTI peptide inhibits trypsin with a submicromolar apparent K i and is expected to be specific for trypsin-like serine proteases. VhTI differs dramatically in structure from all previously described families of trypsin inhibitors, consisting of a helix-turn-helix motif, with the two ␣ helices tightly associated by two disulfide bonds. Unusually, the crystallized complex is in the form of a stabilized acyl-enzyme intermediate with the scissile bond of the VhTI inhibitor cleaved and the resulting N-terminal portion of the inhibitor remaining attached to the trypsin catalytic serine 195 by an ester bond. A synthetic, truncated version of the VhTI peptide has also been produced and co-crystallized with trypsin but, surprisingly, is seen to be uncleaved and consequently forms a noncovalent complex with trypsin. The VhTI peptide shows that effective enzyme inhibitors can be constructed from simple helical motifs and provides a new scaffold on which to base the design of novel serine protease inhibitors.Plant seeds are rich sources of proteinaceous proteinase inhibitors. These are believed to form a wide spectrum defense mechanism against fungal pathogens and invertebrate pests but may also play a role in the regulation of metabolism and act as storage proteins (1). A diverse range of medicinal properties have also been associated with many of these inhibitors, including anti-human immunodeficiency virus activity (2), hemolytic activity (3, 4), and inhibition of neurotensin binding (5) On the basis of their amino acid sequence and target proteinases, plant proteinase inhibitors have been classified into a number of families (6). The two best characterized are the Kunitz and Bowman-Birk families. The Kunitz soybean trypsin inhibitor was the first to be extensively characterized (7) and is an all -sheet protein of 20 kDa. One exposed surface loop and the N terminus of the protein interact closely with the trypsin molecule, whereas the vast majority of the inhibitor forms no direct contacts with its inhibitory target. The Bowman-Birk family of serine protease inhibitors (reviewed in Ref. 8) are smaller proteins of ϳ8 kDa that contain seven conserved disulfide bridges. They have two reactive sites that are able to bind to the active sites of a number of serine proteases including trypsin and chymotrypsin from human, animal, and insect sources. The reactive site residues of the inhibitor lie within a -hairpin region (stabilized by a disulfide bond), which enables them to be presented in the same conformation as the normal peptide substrate (9). Like the Kunitz family of serine protea...
Fluconazole was observed to inhibit sterol 14␣-demethylase in the human pathogen Cryptococcus neoformans, and accumulation of a ketosteroid product was associated with growth arrest. A novel mechanism(s) of azole and amphotericin B cross-resistance was identified, unrelated to changes in sterol biosynthesis, as previously identified in Saccharomyces cerevisiae. Reduced cellular content of drug could account for the resistance phenotype, indicating the possible involvement of a mechanism similar to multidrug resistance observed in higher eukaryotes. Infection withCryptococcus neoformans leading to cryptococcal meningitis has been associated with up to 8% of all AIDS cases (7). Treatment failures and recurrence of infection in AIDS patients on current chemotherapeutic programs involving the use of azole antifungal agents or the polyene antibiotic amphotericin B are increasing (1,3,12,21).Ergosterol is the principal sterol in most fungi (for a review, see reference 9). Amphotericin B has been shown to bind ergosterol in the membrane, with resistance occurring through mutation in the biosynthetic pathway (3). Mode-of-action studies have shown that azole antifungal agents bind to cytochrome P-450 (P-450 14␣-dm ), preventing sterol 14␣-demethylation (20). Subsequent 4-demethylation may still occur, with formation of abnormal 14␣-methyl sterols such as obtusifoliol, 14␣-methylfecosterol, and the presumed product of attempted ⌬ 5(6) desaturation of 14␣-methylfecosterol, 14␣-methyl-3,6-diol (17). The accumulation of this last sterol has been shown genetically and biochemically to be associated with the arrest of growth in azole-treated Saccharomyces cerevisiae (17).Lesions in P-450 14␣-dm confer resistance to polyene antibiotics by preventing the formation of ergosterol, which is the target molecule for polyene action. For viability, these strains require a second defect in sterol ⌬ 5(6) -desaturase which prevents the formation of 14␣-methyl-3,6-diol (14). Azole-resistant mutants generated directly in S. cerevisiae are also defective in sterol ⌬ 5(6) -desaturase (18); these mutants do not accumulate ergosterol and are also cross-resistant to polyene antifungal agents.In the study described in this report we examined the potential for cross-resistance between azole and polyene antifungal agents in the fungal pathogen C. neoformans. A different pattern of sterol accumulation was observed following azole treatment of C. neoformans compared with that observed in S. cerevisiae, suggesting that the mechanisms for avoiding the formation of 14␣-methyl-3,6-diol were not applicable (17). However, mutants which were cross-resistant to the azoles and amphotericin B were isolated, indicating an important consideration for future antifungal therapy. MATERIALS AND METHODSCulture conditions. Two strains of the human pathogen C. neoformans (B4476 and B4500) were obtained from K. J. Kwon-Chung, National Institutes of Health, Bethesda, Md. Growth was supported on 2% (wt/vol) glucose, 2% (wt/vol) Difco peptone, and 1% (wt/vol) Difco yeast e...
Azole antifungals inhibit CYP51Al-mediated sterol 14c~-demethylation and the mechanism(s) of resistance to such compounds in Ustilago maydis were examined. The inhibition of growth was correlated with the accumulation of the snbstrate, 24-methylene-24,25-dihydrolanosterol (eburicol), and depletion of ergosterol. Mutants overcoming the effect of azole antifungal treatment exhibited a unique phenotype with leaky CYP51A1 activity which was resistant to inhibition. The results demonstrate that alterations at the level of inhibitor binding to the target site can produce azole resistance. Similar changes may account for fungal azole resistance phenomena in agriculture, and also in medicine where resistance has become a problem in immunocompromised patients suffering from AIDS.
This article describes the first detailed analysis of mitochondrial electron transfer and oxidative phosphorylation in the pathogenic filamentous fungus, Gaeumannomyces graminis var. tritici. While oxygen consumption was cyanide insensitive, inhibition occurred following treatment with complex III inhibitors and the alternative oxidase inhibitor, salicylhydroxamic acid (SHAM). Similarly, maintenance of a ⌬ across the mitochondrial inner membrane was unaffected by cyanide but sensitive to antimycin A and SHAM when succinate was added as the respiratory substrate. As a result, ATP synthesis through complex V was demonstrated to be sensitive to these two inhibitors but not to cyanide. Analysis of the cytochrome content of mitochondria indicated the presence of those cytochromes normally associated with electron transport in eukaryotic mitochondria together with a third, b-type heme, exhibiting a dithionite-reduced absorbance maxima at 560 nm and not associated with complex III. Antibodies raised to plant alternative oxidase detected the presence of both the monomeric and dimeric forms of this oxidase. Overall this study demonstrates that a novel respiratory chain utilizing the terminal oxidases, cytochrome c oxidase and alternative oxidase, are present and constitutively active in electron transfer in G. graminis tritici. These results are discussed in relation to current understanding of fungal electron transfer and to the possible contribution of alternative redox centers in ATP synthesis.Under aerobic conditions, respiration of carbon metabolites in animal, plant, and fungal cells occurs in a tightly regulated manner to produce carbon dioxide and water. Transfer of the electron pairs associated with the respiration of carbon metabolites is indirect and complex, involving the reduction of the coenzymes NADϩ and FAD at two sites within glycolysis and four sites within the citric acid cycle. The electrons (associated with NADH and FADH 2 ) are subsequently transferred, via at least four distinct sites, into the electron-transport chain, where a series of reduction and oxidation events occur in a sequential manner at approximately 10 different redox centers.In the classical scheme, these redox centers are composed of a series of cytochromes and iron-sulfur complexes. About half the energy generated is lost as heat. The remaining energy generated by the electron flow is utilized in the translocation of hydrogen ions from the mitochondrial matrix to the inter mitochondrial membrane space. The free energy is thus stored in the proton gradient (proton motive force) and is subsequently used to drive the synthesis of ATP.While in mammalian systems the components and sequence of events associated with electron transport appear tightly conserved, those of plants and fungi appear more complex and diverse in nature, often involving alternative redox centers and pathways. Significant research has been conducted in the area of plant respiration, leading to the characterization of these alternative systems, but research into fun...
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