A cDNA, cRKIN1, encoding a putative homologue of the yeast (Saccharomyces cerevisuiae) SNFl (6), human cells (7), and Xenopus (8) are also present in pea (9) and Arabidopsis (10). All protein kinases that have been characterized in detail contain a number of key residues and conserved regions in the catalytic domain (11) but can be divided into two classes: those that phosphorylate serine/threonine residues and those that phosphorylate tyrosine residues. In the present report, we present the nucleotide sequence of a cDNA, cRKIN1,** isolated from a rye endosperm cDNA library. The cRKIN1-encoded protein contains all the invariant residues and conserved domains characteristic ofeukaryotic protein-serine/threonine kinases. It is particularly similar to the product ofthe SNFJ gene of yeast (Saccharomyces cerevisiae) (12), a protein affecting global regulation of carbon metabolism, and the expression of cRKIN1 in yeast snfl mutants restores SNFJ function.

Calcineurin is important for fungal virulence and a potential antifungal target, but compounds targeting calcineurin, such as FK506, are immunosuppressive. Here we report the crystal structures of calcineurin catalytic (CnA) and regulatory (CnB) subunits complexed with FK506 and the FK506-binding protein (FKBP12) from human fungal pathogens (Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans and Coccidioides immitis). Fungal calcineurin complexes are similar to the mammalian complex, but comparison of fungal and human FKBP12 (hFKBP12) reveals conformational differences in the 40s and 80s loops. NMR analysis, molecular dynamic simulations, and mutations of the A. fumigatus CnA/CnB-FK506-FKBP12-complex identify a Phe88 residue, not conserved in hFKBP12, as critical for binding and inhibition of fungal calcineurin. These differences enable us to develop a less immunosuppressive FK506 analog, APX879, with an acetohydrazine substitution of the C22-carbonyl of FK506. APX879 exhibits reduced immunosuppressive activity and retains broad-spectrum antifungal activity and efficacy in a murine model of invasive fungal infection.

The sensitivity of the microaerophilic protozoan Trichomonas vaginalis to oxygen and products of its reduction, and the antioxidant defences employed by this organism, were investigated. Studies revealed that this amitochondrial flagellate is sensitive to oxygen tensions above those experienced in situ in the vagina (i.e. > 60 pM) and that metronidazole-resistant strains (CDC 85 and IR78) were more sensitive to elevated oxygen levels than a metronidazolesensitive isolate (1910). In the presence of radical scavengers, inactivation of organisms at 60 pM oxygen was significantly lessened. Investigation of the antioxidant enzymes present in this organism revealed that activities of peroxide-reducing enzymes (e.g. catalase and general peroxidase) were not detectable, but that a cyanide-insensitive, azide-sensitive superoxide dismutase was present in cell extracts. Measurement of thiol-cycl ing enzymes indicated that NADPH could drive the reduction of oxidized glutathione (thiol reductase) ; however, the corresponding peroxidase activity was not detected. Analysis of thiols in whole cells of T. vaginalis indicated that glutathione was absent, but high levels of other thiols, propanethiol, methanethiol and H,S, were present. No significant differences were detected in thiol levels or antioxidant enzyme activities on comparison of metronidazole-sensitive and resistant strains. These results indicate that the sensitivity of T, vaginalis to oxygen above physiological levels is due to the lack of adequate peroxidereducing enzymes and radical-scavenging mechanisms.

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