Disruption of the Saccharomyces Cerevlsiae gene for NADPH-cytochrome P450 reductase causes increased sensitivity to ketoconazole

Sutter, Thomas R.; Loper, John C.

doi:10.1016/s0006-291x(89)80139-1

Cited by 62 publications

(51 citation statements)

References 13 publications

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“…Therefore, it is most probable that the amino acid substitutions identified in NFI 2013 CaERG11, and that detected by White (1997b), R467K, are in the same way responsible for the observed biochemical insensitivity to fluconazole, and contribute to the overall fluconazole resistance of these strains. However, it remains to be investigated whether underor overexpression of NADPH P450 reductase or cytochrome b & reductase, which alters the susceptibility of S. cerevisiae to azoles (Sutter & Loper, 1989 ;Truan et al, 1994), has a similar effect on the affinity of 14DM for azoles in C. albicans cell extracts, and whether this mode of resistance occurs in Candida isolates. It cannot therefore be concluded without reserve that lower sensitivity of sterol synthesis to azoles in cell-free extracts is invariably associated with mutation(s) of CaERG11.…”

Section: Discussionmentioning

confidence: 99%

Multiple amino acid substitutions in lanosterol 14α-demethylase contribute to azole resistance in Candida albicans

1999

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Lanosterol 14α-demethylase (14DM) is the target of the azole antifungals, and alteration of the 14DM sequence leading to a decreased affinity of the enzyme for azoles is one of several potential mechanisms for resistance to these drugs in Candida albicans. In order to identify such alterations the authors investigated a collection of 19 C. albicans clinical isolates demonstrating either frank resistance (MICs Z32 µg ml N1 ) or dose-dependent resistance (MICs 8-16 µg ml N1 ) to fluconazole. In cell-free extracts from four isolates, including the Darlington strain ATCC 64124, sensitivity of sterol biosynthesis to inhibition by fluconazole was greatly reduced, suggesting that alterations in the activity or affinity of the 14DM could contribute to resistance. Cloning and sequencing of the 14DM gene from these isolates revealed 12 different alterations (two to four per isolate) leading to changes in the deduced amino acid sequence. Five of these mutations have not previously been reported. To demonstrate that these alterations could affect fungal susceptibility to azoles, the 14DM genes from one sensitive and three resistant C. albicans strains were tagged at the carboxyl terminus with a c-myc epitope and expressed in Saccharomyces cerevisiae under control of the endogenous promoter. Transformants receiving 14DM genes from resistant strains had fluconazole MICs up to 32-fold higher than those of transformants receiving 14DM from a sensitive strain, although Western blot analysis indicated that the level of expressed 14DM was similar in all transformants. Amino acid substitutions in the 14DM gene from the Darlington strain also conferred a strong cross-resistance to ketoconazole. In conclusion, multiple genetic alterations in C. albicans 14DM, including several not previously reported, can affect the affinity of the enzyme for azoles and contribute to resistance of clinical isolates.

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Section: Discussionmentioning

confidence: 99%

Multiple amino acid substitutions in lanosterol 14α-demethylase contribute to azole resistance in Candida albicans

1999

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“…CPR1 or NCP1). 43,190 In contrast, ERG25 and ERG3 are thought to depend on the microsomal NADH-cytochrome b5 reductase and cytochrome b5. 18,149 In aerobic cpr1 mutant cells, it has been suggested that NADH-cytochrome b5 reductase S t e r y l e s t e r h y d r o l a s e Figure 3.…”

Section: Oxygen-dependent Sterol Synthesismentioning

confidence: 99%

Role of the non‐respiratory pathways in the utilization of molecular oxygen by Saccharomyces cerevisiae

Rosenfeld

Beauvoit

2003

Yeast

132

110

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Saccharomyces cerevisiae is a facultative anaerobe devoid of mitochondrial alternative oxidase. In this yeast, the structure and biogenesis of the respiratory chain, on the one hand, and the functional interactions of oxidative phosphorylation with the cellular energetic metabolism, on the other, are well documented. However, to our knowledge, the molecular aspects and the physiological roles of the non-respiratory pathways that utilize molecular oxygen have not yet been reviewed. In this paper, we review the various non-respiratory pathways in a global context of utilization of molecular oxygen in S. cerevisiae. The roles of these pathways are examined as a function of environmental conditions, using either physiological, biochemical or molecular data. Special attention is paid to the characterization of the so-called 'cyanide-resistant respiration' that is induced by respiratory deficiency, catabolic repression and oxygen limitation during growth. Finally, several aspects of oxygen sensing are discussed.

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“…For this purpose the plasmids were transformed into the cprlA strain YNW64 which lacked endogenous yeast oxidoreductase due to a disruption of the CPR1 gene. Such crplA strains are viable on normal media but they react supersensitively towards the antifungal drug ketoconazole (24). The supersensitive phenotype is rescued by expression of human oxidoreductase cDNA in such strains (18).…”

Section: Co-expression Of Cytochrome P-450 and Oxidoreductase Cdnas Imentioning

confidence: 99%

High promutagen activating capacity of yeast microsomes containing human cytochrome P-450 1A and human NADPH-cytochrome P-450 reductase

1994

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Yeast Saccharomyces cerevisiae strains have been constructed that co-express cDNAs coding for the human cytochrome P-450 enzymes CYP1A1 or CYP1A2 in combination with human NADPH-cytochrome P-450 reductase (oxidoreductase). Microsomal fractions prepared from the strains were able to efficiently activate various drugs to Salmonella mutagens. These experiments demonstrated that a functional interaction occurred between the respective human enzymes in the yeast microsomes. For every drug tested, the microsomes containing CYP enzymes and oxidoreductase were 2-to 4-fold better in activation than the corresponding microsomes that contained CYP alone. Interestingly, co-expression of CYP1A2 with oxidoreductase resulted in a decrease of 7-ethoxyresorufin-0-deethylase activity, a problem which is related to this specific substrate. Using the microsomes, it was demonstrated that aflatoxin B, was activated to a mutagen not only by CYP1A2 but also by CYP1A1. In contrast, benzo[a]pyrene was exclusively activated by CYP1A1 whereas CYP1A2 was inactive. The drug 3-amino-l-methyl-5//-pyrido[4,3-ft]indole (Trp-P-2) was activated by CYP1A2 and to a lesser extent by CYP1A1. A strong substrate specificity was observed with the two structurally related heterocyclic arylamines 2-amino-3,4-dimethyUmidazo[4,5-/]quinoUne (MelQ) and 2-amino-3,8-dimethyUmidazo[4,5-/]quinoxaline (MelQ*). MeIQ x was activated efficiently by both CYP enzymes, whereas MelQ was only activated by CYP1A2 and not by CYP1A1. The fact that microsomes from vector transformed control strains were unable to activate any of the drugs studied underlines the suitability of these microsomes for metabolic studies. Moreover, the presence of suitable marker genes in the yeast strains will enable us to study mitotic recombination and gene conversion events induced by drugs that require metabolic activation.

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Disruption of the Saccharomyces Cerevlsiae gene for NADPH-cytochrome P450 reductase causes increased sensitivity to ketoconazole

Cited by 62 publications

References 13 publications

Multiple amino acid substitutions in lanosterol 14α-demethylase contribute to azole resistance in Candida albicans

Multiple amino acid substitutions in lanosterol 14α-demethylase contribute to azole resistance in Candida albicans

Role of the non‐respiratory pathways in the utilization of molecular oxygen by Saccharomyces cerevisiae

High promutagen activating capacity of yeast microsomes containing human cytochrome P-450 1A and human NADPH-cytochrome P-450 reductase

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