Partial purification of pisatin demethylase, a cytochrome P-450 from the pathogenic fungus Nectria haematococca

1986 ◽  
Vol 144 (1) ◽  
pp. 84-90 ◽  
Author(s):  
Anne E. Desjardins ◽  
Hans D. VanEtten
Keyword(s):  
Pathogenic Fungus ◽  
Partial Purification ◽  
Nectria Haematococca ◽  
Pisatin Demethylase ◽  
Cytochrome P 450

scholarly journals Stereoselective interaction of the azole antifungal agent SCH39304 with the cytochrome P-450 monooxygenase system isolated from Cryptococcus neoformans.

1997 ◽  
Vol 41 (7) ◽  
pp. 1465-1467 ◽  
Author(s):  
D C Lamb ◽  
B C Baldwin ◽  
K J Kwon-Chung ◽  
S L Kelly
Keyword(s):  
Cryptococcus Neoformans ◽  
Pathogenic Fungus ◽  
Ergosterol Biosynthesis ◽  
Classical Type ◽  
Monooxygenase System ◽  
Inhibition Of Growth ◽  
Fluconazole Therapy ◽  
Specific Localization ◽  
Cytochrome P 450

We investigated the stereoselective inhibition of growth and ergosterol biosynthesis by SCH39304 in the pathogenic fungus Cryptococcus neoformans obtained from four AIDS patients who failed fluconazole therapy and compared the results to those obtained with a wild-type strain. For all strains, the MICs of the RR isomer were approximately half those of the racemate, with the SS enantiomer showing no inhibitory activity. The 50% inhibitory concentrations for in vitro ergosterol biosynthesis correlated with the MIC data, indicating stereoselective inhibition of their target P-450 enzyme, sterol 14alpha-demethylase, as the cause of this difference. The RR enantiomer produced classical type II spectra on addition to microsomal extracts of the strains, whereas the SS enantiomer showed an absence of binding. Stereo- and regio-specific localization of N-1 substituent groups of SCH39304 within the active site of the enzyme determined the unique discrimination between its two enantiomers, and the inability to bind to sterol 14alpha-demethylase is also true of other P-450 enzymes contained in the microsomal fraction. As previously observed for other antifungal azoles, isolates obtained following failure of fluconazole therapy showed resistance to SCH39304 and its RR enantiomer. This resistance could be associated with an alteration in the sensitivity of ergosterol biosynthesis in vitro. These alterations did not cause any changes allowing the SS enantiomer to bind to the P-450 mediating sterol 14alpha-demethylation.

scholarly journals Purification, Reconstitution, and Inhibition of Cytochrome P-450 Sterol Δ22-Desaturase from the Pathogenic Fungus Candida glabrata

1999 ◽  
Vol 43 (7) ◽  
pp. 1725-1728 ◽  
Author(s):  
David C. Lamb ◽  
Segula Maspahy ◽  
Diane E. Kelly ◽  
Nigel J. Manning ◽  
Antonia Geber ◽  
...  
Keyword(s):  
Candida Glabrata ◽  
Desaturase Activity ◽  
Pathogenic Fungus ◽  
Kinetic Studies ◽  
Gene Encoding ◽  
Fungal Cell ◽  
Total Inhibition ◽  
Reconstituted System ◽  
Cytochrome P 450 ◽  
Genome Projects

ABSTRACT Sterol Δ22-desaturase has been purified from a strain of Candida glabrata with a disruption in the gene encoding sterol 14α-demethylase (cytochrome P-45051; CYP51). The purified cytochrome P-450 exhibited sterol Δ22-desaturase activity in a reconstituted system with NADPH–cytochrome P-450 reductase in dilaurylphosphatidylcholine, with the enzyme kinetic studies revealing a Km for ergosta-5,7-dienol of 12.5 μM and aV max of 0.59 nmol of this substrate metabolized/min/nmol of P-450. This enzyme is encoded by CYP61 (ERG5) in Saccharomyces cerevisiae, and homologues have been shown in the Candida albicans andSchizosaccharomyces pombe genome projects. Ketoconazole, itraconazole, and fluconazole formed low-spin complexes with the ferric cytochrome and exhibited type II spectra, which are indicative of an interaction between the azole moiety and the cytochrome heme. The azole antifungal compounds inhibited reconstituted sterol Δ22-desaturase activity by binding to the cytochrome with a one-to-one stoichiometry, with total inhibition of enzyme activity occurring when equimolar amounts of azole and cytochrome P-450 were added. These results reveal the potential for sterol Δ22-desaturase to be an antifungal target and to contribute to the binding of drugs within the fungal cell.

scholarly journals Ring-and N-hydroxylation of 2-acetamidofluorene by rat liver reconstituted cytochrome P-450 enzyme system

1975 ◽  
Vol 150 (3) ◽  
pp. 561-564 ◽  
Author(s):  
P D Lotlikar ◽  
K Zaleski
Keyword(s):  
Cytochrome C ◽  
Rat Liver ◽  
Enzyme System ◽  
Partial Purification ◽  
Hydroxylated Metabolites ◽  
Triton X ◽  
Cytochrome P 450 ◽  
Ring Hydroxylation ◽  
Hydroxylation Activity ◽  
Nadph Cytochrome C Reductase

The N- and ring-hydroxylation of 2-acetamidofluorene were studied with a reconstituted cytochrome P-450 enzyme from microsomal fractions of liver from both control and 3-methylcholanthrene-pretreated rats. Proteinase treatment and Triton X-100 solubilization were two important steps for partial purification of the cytochrome P-450 fraction. Both cytochrome P-450 and NADPH-cytochrome c reductase fractions were required for optimum N- and ring-hydroxylation activity. Hydroxylation activity was determined by the source of cytochrome P-450 fraction; cytochrome P-450 fraction from pretreated animals was severalfold more active than the fraction from controls. Formation of N-hydroxylated metabolites with reconstituted systems from both control and pretreated animals was greater than that with their respective whole microsomal fractions.

Partial purification of human liver cytochrome P 450

1979 ◽  
Vol 88 (3) ◽  
pp. 826-832 ◽  
Author(s):  
Ph. Beaune ◽  
P. Dansette ◽  
J.P. Flinois ◽  
S. Columelli ◽  
D. Mansuy ◽  
...  
Keyword(s):  
Human Liver ◽  
Partial Purification ◽  
Liver Cytochrome ◽  
Cytochrome P 450
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