The mutation T315A in Candida albicans sterol 14alpha-demethylase causes reduced enzyme activity and fluconazole resistance through reduced affinity.

Sterol 14alpha-demethylase (P45051) is the target for azole antifungal compounds, and resistance to these drugs and agrochemicals is of significant practical importance. We undertook site-directed mutagenesis of the Candida albicans P45051 heterologously expressed in Saccharomyces cerevisiae to probe a model structure for the enzyme. The change T315A reduced enzyme activity 2-fold as predicted for the removal of the residue that formed a hydrogen bond with the 3-OH of the sterol substrate and helped to locate it in the active site. This alteration perturbed the heme environment, causing an altered reduced carbon monoxide difference spectrum with a maximum at 445 nm. The changes also reduced the affinity of the enzyme for the azole antifungals ketoconazole and fluconazole and after expression induced by galactose caused 4-5-fold azole resistance in transformants of S. cerevisiae. This is the first example of a single base change in the target enzyme conferring resistance to azoles through reduced azole affinity.

The mechanism of the acyl-carbon bond cleavage reaction catalyzed by recombinant sterol 14 alpha-demethylase of Candida albicans (other names are: lanosterol 14 alpha-demethylase, P-45014DM, and CYP51).

The Candida albicans sterol 14 alpha-demethylase gene (P-45014DM, CYP51) was transferred to the yeast plasmid YEp51 placing it under the control of the GAL10 promoter. The resulting construct (YEp51:CYP51) when transformed into the yeast strain GRF18 gave a clone producing 1.5 mu mol of P-450/liter of culture, the microsomal fraction of which contained up to 2.5 nmol of P-450/mg of protein. Two oxygenated precursors for the 14 alpha-demethylase, 3 beta-hydroxylanost-7-en-32-al and 3 beta-hydroxylanost-7-en-32-ol, variously labeled with 2H and 18O at C-32 were synthesized. In this study the conversion of [32-2H,32-16O]- and [32-2H,32-18O]3 beta-hydroxylanost-7-en-32-al with the recombinant 14 alpha-demethylase was performed under 16O2 or 18O2 and the released formic acid analyzed by mass spectrometry. The results showed that in the acyl-carbon bond cleavage step (i.e. the deformylation process) the original carbonyl oxygen at C-32 of the precursor is retained in formic acid and the second oxygen of formate is derived from molecular oxygen; precisely the same scenario that has previously been observed for the acyl-carbon cleavage steps catalyzed by aromatase (P-450arom) and 17 alpha-hydroxylase-17,20-lyase (P-45017 alpha,CYP17). In the light of these results the mechanism of the acyl-carbon bond cleavage step catalyzed by the 14 alpha-demethylase is considered.

Mechanistic kinship between hydroxylation and desaturation reactions: acyl-carbon bond cleavage promoted by pig and human CYP17 (P-450(17)alpha; 17 alpha-hydroxylase-17,20-lyase).

Using homogeneous pig and recombinant human CYP17, the mechanism of the acyl-carbon bond fission involved in the direct cleavage of pregnenolone was studied. It was found that the formation of androsta-5,16-dien-3 beta-ol (5,16-diene) and androst-5-ene-3 beta,17 alpha-diol (17 alpha-hydroxyandrogen) from pregnenolone was catalyzed by both the isoforms and that the two conversions were dependent on the presence of cytochrome b5 (cyt b5). 3 beta-Hydroxyandrost-5-ene-17 beta-carbaldehyde (aldehyde), an analogue of the physiological substrate pregnenolone, was handled as a substrate by both isoforms of CYP17. The aldehyde underwent cleavage to produce the 5,16-diene plus the 17 alpha-hydroxyandrogen, at rates approximately 8- and 3-fold higher than any physiological reaction catalyzed, in the absence of cytochrome b5, by the pig and human CYP17 isoforms, respectively. The stereochemistry of the reaction was studied using the aldehyde labeled with 2H at three strategic positions, 16 alpha, 16 beta, and 17 alpha, with incubations performed under both 16O2 and 18O2. The results showed that the formation of the 5,16-diene is attended by the removal of the 16 alpha-hydrogen atom; all three 2H atoms are retained in the formation of 17 alpha-hydroxyandrogen and its 17 alpha-hydroxyl oxygen originates from O2. Irrespective of the nature of the substrate, or the enzymic conditions used, the 5,16-diene and 17 alpha-hydroxyandrogen were produced in similar ratios, suggesting that their genesis is closely linked. Both the compounds may be envisaged to arise from a peroxy adduct that fragments to give a carbon radical that then undergoes either a disproportionation or an oxygen-rebound reaction. The conclusion was supported by isotope-partitioning experiments when the conversion of a mixture of the unlabeled aldehyde and its isotopomer, containing 2H at 16 alpha as well as 16 beta, led to the enrichment of 2H in 17 alpha-hydroxyandrogen. It is suggested that the mechanistic kinship between hydroxylation and olefin formation, revealed by the present study, also applies to conventional hydroxylation and desaturation reactions.

Mechanism of the acyl-carbon cleavage and related reactions catalyzed by multifunctional P-450s: studies on cytochrome P-450(17)alpha.

It is now well-known that conventional cytochrome P-450s catalyze hydroxylation reactions using an iron mono-oxygen species, the structure of which, as inferred from chemical model studies, may be drrepresented by the following canonical forms: FeV==O<-->(.+)FeIV==O<-->FeIV--O(.). Certain multifunctional P-450s, notably those involved in steroid biosynthesis, catalyze, in addition to hydroxylation reactions, an acyl-carbon cleavage process in which the participation of an iron peroxide intermediate, FeIII--OOH, has been suggested. However the possibility still exists that the C--C bond cleavage may also occur using the FeV==O species. We have scrutinized the chemical consequences of involving either an FeV==O or an FeIII--OOH species for five different C--C bond cleavage reactions. With respect to the status as well as the origin of hydrogen and oxygen atoms, in four of the examples the mechanism involving the FeV==O species makes the same prediction as that using the iron peroxide intermediate, that is, the incorporation of an atom of oxygen from O2 into acyl part of the cleaved fragment. The fifth example, however, involving the formation, with pig testes microsomes, of 17 alpha-hydroxyandrogen (androst-5-ene-3 beta,17 alpha-diol) from pregnenolone, presents an interesting contrast--in this case different outcomes are predicted by the two mechanisms. These possibilities have been experimentally evaluated using substrates stereo- and regiospecifically labeled with heavy isotopes and incubated with pig testes microsomes under either 16O2 or 18O2.(ABSTRACT TRUNCATED AT 250 WORDS)

Investigations of ibuprofen and paracetamol binding to lens proteins to explore their protective role against cataract.

There is evidence that ibuprofen and paracetamol can act as anti-cataract drugs. [14C]-Ibuprofen labelled at the methyl group of the propanoic acid moiety was synthesized. The labelled ibuprofen was found to bind non-covalently to alpha-crystallin but not to beta- and gamma-crystallin of the bovine lens. Labelled paracetamol binds to total lens soluble proteins. Both drugs penetrate into the lens cortex and nucleus within 24 hr. Affinity chromatographic studies suggest that the lipophilic isobutyl group of ibuprofen hinders binding to the lens proteins. Hence, in the light of weak binding of ibuprofen and paracetamol and strong binding of the ibuprofen analogue used in the affinity chromatography, it is suggested, in this paper, that the protection against cataract by these analgesics is possibly due to their metabolites interacting with the lens proteins.