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Trichothecene resistance is achieved via 3-O-acetylation on the biosynthetic pathway of deoxynivalenol in Fusarium graminearum. The responsible 3-O-acetyltransferase gene, Tri101, was located between the UTP-ammonia ligase gene and the phosphate permease gene, and not in thetrichothecene biosynthetic gene cluster. As predicted by the presence of its homologues in yeasts, the resistance gene proved to have evolved independently of other biosynthetic genes. In Fusarium sporotrichioides, FsTri101 (a functional homologue of Tri101) was also locatedthese two 'house-keeping' genes. However, FsTri101 appeared not to play a pivotal role for self-resistance, suggesting that other defensive options are the primary working strategies for the type A trichothecene producer. (c) 1999 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

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 alpha 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.

Malaria caused by Plasmodium vivax is a major cause of global morbidity and, in rare cases, mortality. Lactate dehydrogenase is an essential Plasmodium protein and, therefore, a potential antimalarial drug target. Ideally, drugs directed against this target would be effective against both major species of Plasmodium, P. falciparum and P. vivax. In this study, the crystal structure of the lactate dehydrogenase protein from P. vivax has been solved and is compared to the equivalent structure from the P. falciparum enzyme. The active sites and cofactor binding pockets of both enzymes are found to be highly similar and differentiate these enzymes from their human counterparts. These structures suggest effective inhibition of both enzymes should be readily achievable with a common inhibitor. The crystal structures of both enzymes have also been solved in complex with the synthetic cofactor APADH. The unusual cofactor binding site in these Plasmodium enzymes is found to readily accommodate both NADH and APADH, explaining why the Plasmodium enzymes retain enzymatic activity in the presence of this synthetic cofactor.

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.

This paper reviews the current status of our understanding of azole antifungal resistance mechanisms at the molecular level and explores their implications. Extensive biochemical studies have highlighted a significant diversity in mechanisms conferring resistance to azoles, which include alterations in sterol biosynthesis, target site, uptake and efflux. In stark contrast, few examples document the molecular basis of azole resistance. Those that do refer almost exclusively to mechanisms in laboratory mutants, with the exception of the role of multi-drug resistance proteins in clinical isolates of Candida albicans. It is clear that the technologies required to examine and define azole resistance mechanisms at the molecular level exist, but research appears distinctly lacking in this most important area.

Resistance to azole antifungals in Ustilago maydis was associated with a leaky defect in sterol delta 5(6)desaturase. This defect resulted in reduced accumulation of 14 alpha-methylergosta-24(28)-diene-3 beta,6 alpha-diol and an increase in the proportion of 14 alpha-methylfecosterol in treated cells when compared to the parent strain. The results demonstrate the importance of this mechanism in pathogenic fungi.