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Two Cryptococcus neoformans strains isolated from an AIDS patient were investigated, a pretreatment isolate (CN1) and a second isolate (CN3) following failure of fluconazole and amphotericin B treatment. No difference in fluconazole sensitivity, but relative resistance to amphotericin B was observed for CN3. The sterol composition of CN3 indicated a defect in sterol delta 8-->7 isomerase in this strain and depletion of ergosterol, the major sterol of the CN1.

Cytochrome P-45061 (CYP61) was a cytochrome P-450 revealed during the yeast genome project when chromosome XIII was sequenced. Here we report on the properties of this second microsomal P-450 of vegetatively growing yeast. The enzyme kinetics associated with its endogenous role in sterol ⌬ 22-desaturation revealed a K m of 20.4 M and a V max of 2.9nmol/min/nmol CYP61. The affinity of the enzyme for antifungal drugs was characterized to investigate its potential role in determining tolerance to these sterol 14␣-demethylase (CYP51) inhibitors. Drug binding induced a type II spectral change, which became saturated at equimolar concentrations of azole drug and P-450. Fluconazole exhibited slightly reduced affinity in comparison to ketoconazole as indicated by carbon monoxide displacement. These and K i determination for fluconazole (0.14 nM) revealed CYP61 to have a similar affinity to azole drugs when compared with data available for CYP51, and the implications for antifungal treatment were considered.The cytochrome P-450 (CYP) superfamily are involved in a variety of monooxygenase reactions including xenobiotic and endogenous substrates. Genome projects are uncovering more genes encoding such proteins, and one such instance was cyp61 (ERG5) encoded on chromosome XIII of Saccharomyces cerevisiae (1). Previous studies on S. cerevisiae have identified a single form of P-450 during purification from microsomal fractions of vegetatively growing yeast. This P-450 performed a role in sterol 14␣-demethylation (2), later associated with the gene, cyp51 (ERG11; Ref. 3). This is the only P-450 activity associated with a family found in plants, animals, and fungi (4). Other studies based on inhibition and co-factor characteristics indicated a further P-450-mediated activity was present in vegetative yeast (5). This was confirmed when such a protein was purified from microsomes obtained from a vegetatively growing strain with a cyp51 gene disruption (6). N-terminal amino acid sequence confirmed the protein to be CYP61 from chromosome XIII (1).Inhibitors of CYP51 are of considerable commercial importance as antifungal compounds and selectively inhibit fungal CYP51 over the mammalian and plant counterparts (7). Because they may also bind to CYP61, a potential antifungal target, we characterized the affinity of this P-450 for two of the main drugs employed, ketoconazole and fluconazole, to consider the relative potency and potential contribution CYP61 may make to azole antifungal susceptibility. EXPERIMENTAL PROCEDURESMaterials-Unless specified, all chemicals were obtained from Sigma (Poole, Dorset, UK). Ketoconazole was purchased from Janssen Pharmaceutica, and fluconazole was from Pfizer. Microsomes were prepared, and CYP61 was purified from semi-anaerobically grown cells of the S. cerevisiae strain DK2, which contains a gene disruption in cyp51 (⌬cyp51; ⌬erg11) as described previously (6). Rabbit NADPH-cytochrome P-450 reductase was a gift from Prof. M. Akhtar, University of Southampton. Ergosta-5,7-dienol was purified from a ...

P450 was purified from microsomal fractions of a strain of Saccharomyces cerevisiae which contained detectable P450 despite the disruption of CYP51AI. The P450 had a molecular mass of 58 kDa, similar to P450 51A1, and in a reconstituted assay with rabbit NADPH-P450 rednctase and dilauryl phosphotidylcholine exhibited activity for conversion of ergosta-5,7-dienol into ergosterol. N-Terminal amino acid sequencing of the purified protein corresponded to the translated sequence of P450 61 which was recently identified during sequencing of chromosome XIII. This allowed the function of this family of P450 to be identified as sterol AZ2-desaturation in the pathway of ergosterol biosynthesis. K, v words:Sterol A:2-desaturase; Purification; P450 61In this study we undertook the purification of the residual P450 of vegetative yeast from the strain lacking sterol 14~-demethylase, examined its spectral properties and reconstituted activity. This enabled the identification of its sterol 22-desaturase activity and allowed the function of the recently identified P450 61 family to be established. Materials and methods StrainsSaccharomyces cerevisiae strain DK2 was used [4] as the source of microsomal P450. It is a haploid strain containing a gene disrupted sterol 14~-demethylase (ACYP51A1; ergll) and a suppressor mutation in sterol AS'6-desaturase (erg3) which allows the sterol 14ct-demethylase disruptant to grow aerobically by changing the sterol accumulating to 14~-methylfecosterol [4]. Additional auxotrophic markers include/eu 23,2-112 trp 1~89 his3-A1.

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