scholarly journals Solubilization and Reconstitution of Pisatin Demethylase, a Cytochrome P-450 from the Pathogenic Fungus Nectria haematococca

1984 ◽  
Vol 75 (3) ◽  
pp. 611-616 ◽  
Author(s):  
Anne E. Desjardins ◽  
David E. Matthews ◽  
Hans D. Vanetten
Keyword(s):  
Pathogenic Fungus ◽  
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 Pisatin Demethylase Genes Are on Dispensable Chromosomes While Genes for Pathogenicity on Carrot and Ripe Tomato Are on Other Chromosomes in Nectria haematococca

2002 ◽  
Vol 15 (8) ◽  
pp. 840-846 ◽  
Author(s):  
Deanna L. Funnell ◽  
Hans D. VanEtten
Keyword(s):  
Virulence Genes ◽  
The Other ◽  
Homogeneous Electric Field ◽  
Nectria Haematococca ◽  
Significant Similarity ◽  
Pisatin Demethylase ◽  
Dispensable Chromosome ◽  
Significant Difference ◽  
Ripe Tomato ◽  
Specific Virulence

Studies on the wide-host-range fungus Nectria haematococca MP VI have shown a linkage between virulence on pea and five of nine PDA genes that encode the ability to detoxify the pea phytoalexin, pisatin. Most of the PDA genes are on chromosomes of approximately 1.6 megabases (Mb) and two of these genes, PDA1-2 and PDA6-1, have been demonstrated to reside on approximately 1.6-Mb chromosomes that can be lost during meiosis. Prior studies also have shown that the dispensable chromosome carrying PDA6-1 contains a gene (MAK1) necessary for maximum virulence on chickpea. The present study evaluated whether the other approximately 1.6-Mb chromosomes that carry PDA genes also are dispensable, their relationship to each other, and whether they contain genes for pathogenicity on hosts other than pea or chickpea. DNA from the PDA1-1 chromosome (associated with virulence on pea) and the PDA6-1 chromosome (associated with virulence on chickpea) were used to probe blots of contour-clamped homogeneous electric field (CHEF) gels of isolates carrying different PDA genes and genetically related Pda¯ isolates. All of the approximately 1.6-Mb PDA-bearing chromosomes hybridized with both probes, indicating that they share significant similarity. Genetically related Pda¯ progeny lacked chromosomes of approximately 1.6 Mb and there was no significant hybridization of any chromosomes to the PDA1-1 and PDA6-1 chromosome probes. When isolates carrying different PDA genes and related Pda¯ isolates were tested for virulence on carrot and ripe tomato, there was no significant difference in lesion sizes produced by Pda+ and Pda- isolates, indicating that genes for pathogenicity on these hosts are not on the PDA-containing chromosomes. These results support the hypothesis that the chromosomes carrying PDA genes are dispensable and carry host-specific virulence genes while genes for pathogenicity on other hosts are carried on other chromosomes.

scholarly journals Physical Map of a Conditionally Dispensable Chromosome in Nectria haematococca Mating Population VI and Location of Chromosome Breakpoints

Genetics ◽  
2000 ◽  
Vol 155 (3) ◽  
pp. 1083-1094 ◽  
Author(s):  
Jürg Enkerli ◽  
Heather Reed ◽  
Angela Briley ◽  
Garima Bhatt ◽  
Sarah F Covert
Keyword(s):  
Dna Sequences ◽  
Physical Map ◽  
Pathogenic Fungus ◽  
Restriction Enzymes ◽  
Nectria Haematococca ◽  
Map Construction ◽  
Dispensable Chromosome ◽  
Mating Population ◽  
Polymerase Chain ◽  
Dispersed Repeats

Abstract Certain isolates of the plant pathogenic fungus Nectria haematococca mating population (MP) VI contain a 1.6-Mb conditionally dispensable (CD) chromosome carrying the phytoalexin detoxification genes MAK1 and PDA6-1. This chromosome is structurally unstable during sexual reproduction. As a first step in our analysis of the mechanisms underlying this chromosomal instability, hybridization between overlapping cosmid clones was used to construct a map of the MAK1 PDA6-1 chromosome. The map consists of 33 probes that are linked by 199 cosmid clones. The polymerase chain reaction and Southern analysis of N. haematococca MP VI DNA digested with infrequently cutting restriction enzymes were used to close gaps and order the hybridization-derived contigs. Hybridization to a probe extended from telomeric repeats was used to anchor the ends of the map to the actual chromosome ends. The resulting map is estimated to cover 95% of the MAK1 PDA6-1 chromosome and is composed of two ordered contigs. Thirty-eight percent of the clones in the minimal map are known to contain repeated DNA sequences. Three dispersed repeats were cloned during map construction; each is present in five to seven copies on the chromosome. The cosmid clones representing the map were probed with deleted forms of the CD chromosome and the results were integrated into the map. This allowed the identification of chromosome breakpoints and deletions.

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