Candida albicans NADPH-P450 reductase: Expression, purification, and characterization of recombinant protein

Park, Hyoung-Goo; Lim, Young-Ran; Eun, Chang-Yong; Han, Songhee; Han, Jung‐Soo; Cho, Kyoung Sang; Chun, Young‐Jin; Kim, Donghak

doi:10.1016/j.bbrc.2010.04.138

Cited by 29 publications

(17 citation statements)

References 24 publications

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“…This was reflected in the pale green color of AfCPR1 instead of the yellow color of the fully oxidized form previously obtained for truncated S. cerevisiae CPR (24). The spectral intensity of the second flavin peak (ϳ465 nm) of AfCPR1 fell during reduction with 4, 40, and 200 M NADPH, as previously observed for H. sapiens CPR (23), Phanerochaete chrysosporium CPR (25), and C. albicans CPR (26). The blue semiquinone peak at ϳ590 nm also fell in intensity at the higher NADPH concentrations of 40 and 200 M (Fig.…”

Section: Cpr Phylogenicitysupporting

confidence: 75%

In Vitro Biochemical Study of CYP51-Mediated Azole Resistance in Aspergillus fumigatus

Warrilow

Parker

Price

et al. 2015

Antimicrob Agents Chemother

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bThe incidence of triazole-resistant Aspergillus infections is increasing worldwide, often mediated through mutations in the CYP51A amino acid sequence. New classes of azole-based drugs are required to combat the increasing resistance to existing triazole therapeutics. In this study, a CYP51 reconstitution assay is described consisting of eburicol, purified recombinant Aspergillus fumigatus CPR1 (AfCPR1), and Escherichia coli membrane suspensions containing recombinant A. fumigatus CYP51 proteins, allowing in vitro screening of azole antifungals. Azole-CYP51 studies determining the 50% inhibitory concentration (IC 50 ) showed that A. fumigatus CYP51B (Af51B IC 50 , 0.50 M) was 34-fold more susceptible to inhibition by fluconazole than A. fumigatus CYP51A (Af51A IC 50 , 17 M) and that Af51A and Af51B were equally susceptible to inhibition by voriconazole, itraconazole, and posaconazole (IC 50 s of 0.16 to 0.38 M). Af51A-G54W and Af51A-M220K enzymes were 11-and 15-fold less susceptible to inhibition by itraconazole and 30-and 8-fold less susceptible to inhibition by posaconazole than wild-type Af51A, confirming the azole-resistant phenotype of these two Af51A mutations. Susceptibility to voriconazole of Af51A-G54W and Af51A-M220K was only marginally lower than that of wild-type Af51A. Susceptibility of Af51A-L98H to inhibition by voriconazole, itraconazole, and posaconazole was only marginally lower (less than 2-fold) than that of wild-type Af51A. However, Af51A-L98H retained 5 to 8% residual activity in the presence of 32 M triazole, which could confer azole resistance in A. fumigatus strains that harbor the Af51A-L98H mutation. The AfCPR1/Af51 assay system demonstrated the biochemical basis for the increased azole resistance of A. fumigatus strains harboring G54W, L98H, and M220K Af51A point mutations.

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Section: Cpr Phylogenicitysupporting

confidence: 75%

In Vitro Biochemical Study of CYP51-Mediated Azole Resistance in Aspergillus fumigatus

Warrilow

Parker

Price

et al. 2015

Antimicrob Agents Chemother

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“…An alignment of these 12 CPR proteins ( Fig. 1) indicated that conservation among the FMN, FAD, and NADPH binding regions (20) was variable. The FAD-2 binding region was the most highly conserved, with 7 out of 10 residues being conserved, and the FAD-3 binding region was the least conserved (1 out of 5 residues).…”

Section: Resultsmentioning

confidence: 99%

Novel Substrate Specificity and Temperature-Sensitive Activity of Mycosphaerella graminicola CYP51 Supported by the Native NADPH Cytochrome P450 Reductase

Price

Warrilow

Parker

et al. 2015

Appl Environ Microbiol

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bMycosphaerella graminicola (Zymoseptoria tritici) is an ascomycete filamentous fungus that causes Septoria leaf blotch in wheat crops. In Europe the most widely used fungicides for this major disease are demethylation inhibitors (DMIs). Their target is the essential sterol 14␣-demethylase (CYP51), which requires cytochrome P450 reductase (CPR) as its redox partner for functional activity. The M. graminicola CPR (MgCPR) is able to catalyze the sterol 14␣-demethylation of eburicol and lanosterol when partnered with Candida albicans CYP51 (CaCYP51) and that of eburicol only with M. graminicola CYP51 (MgCYP51). The availability of the functional in vivo redox partner enabled the in vitro catalytic activity of MgCYP51 to be demonstrated for the first time. MgCYP51 50% inhibitory concentration (IC 50 ) studies with epoxiconazole, tebuconazole, triadimenol, and prothioconazole-desthio confirmed that MgCYP51 bound these azole inhibitors tightly. The characterization of the MgCPR/MgCYP51 redox pairing has produced a functional method to evaluate the effects of agricultural azole fungicides, has demonstrated eburicol specificity in the activity observed, and supports the conclusion that prothioconazole is a profungicide. Mycosphaerella graminicola (Zymoseptoria tritici) is an ascomycete filamentous fungus and is the causative pathogen of Septoria leaf blotch in wheat crops. This is the most common cereal crop disease in Europe and can result in devastating losses in crop yield (1, 2). Currently the most widely used fungicides for the control of M. graminicola are demethylase inhibitors (DMIs), which bind to the target cytochrome P450 enzyme, CYP51 (ERG11).CYP51s are ubiquitous in nature and mediate the essential sterol 14␣-demethylation step in the sterol biosynthetic pathway (3). The 14␣-demethylated products are precursors leading to formation of cholesterol (mammals) and ergosterol (fungi) and the formation of a variety of 24-alkylated and desaturated sterols in algae, plants, and protozoa (3). Cholesterol, ergosterol, and sitosterols (plants) play an important structural role in regulating membrane fluidity and permeability of plasma membranes and indirectly modulate the distribution and activity of membrane proteins and ion channels (3, 4). In addition, sterols are precursors of steroid hormones in mammals, brassinosteroids in plants, and ecdysteroids in insects. In yeast and fungi, the CYP51 enzymes are synonymous with lanosterol and eburicol 14␣-demethylation in the production of ergosterol, but as this study shows, M. graminicola CYP51 is able to demethylate only eburicol, in the presence of its native reductase partner, exhibiting novel substrate specificity.The introduction of new azole antifungal compounds has allowed control of M. graminicola infections in wheat to be maintained despite increased tolerance/resistance. The most recently introduced azole is the triazolinethione derivative prothioconazole. However, the control of this disease has been threatened by the identification of mutations in the CYP51 en...

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“…A functional heme oxygenase from C. albicans , the product of the CaHmx1 gene, has also been characterized [20]. Recently, the NPR of C. albicans , coded by the NCP1 gene, was characterized [31]. The purified C. albicans NPR was shown to be an orthologous reductase that supports both the cytochrome P450 and heme oxygenase activities.…”

Section: Discussionmentioning

confidence: 99%

Heterologous expression and characterization of the sterol 14α-demethylase CYP51F1 from Candida albicans

Park

Lee

Chun

et al. 2011

Archives of Biochemistry and Biophysics

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Lanosterol 14α-demethylase (CYP51F1) from Candida albicans is known to be an essential enzyme in fungal sterol biosynthesis. Wild-type CYP51F1 and several of its mutants were heterologously expressed in Escherichia coli, purified, and characterized. It exhibited a typical reduced CO-difference spectrum with a maximum at 446 nm. Reconstitution of CYP51F1 with NADPH-P450 reductase gave a system that successfully converted lanosterol to its demethylated product. Titration of the purified enzyme with lanosterol produced a typical type I spectral change with Kd = 6.7 μM. The azole antifungal agents econazole, fluconazole, ketoconazole, and itraconazole bound tightly to CYP51F1 with Kd values between 0.06 – 0.42 μM. The CYP51F1 mutations F105L, D116E, Y132H, and R467K frequently identified in clinical isolates were examined to determine their effect on azole drug binding affinity. The azole Kd values of the purified F105L, D116E, and R467K mutants were little altered. A homology model of C. albicans CYP51F1 suggested that Tyr132 in the BC loop is located close to the heme in the active site, providing a rationale for the modified heme environment caused by the Y132H substitution. Taken together, functional expression and characterization of CYP51F1 provide a starting basis for design of agents effective against C. albicans infections.

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Candida albicans NADPH-P450 reductase: Expression, purification, and characterization of recombinant protein

Cited by 29 publications

References 24 publications

In Vitro Biochemical Study of CYP51-Mediated Azole Resistance in Aspergillus fumigatus

In Vitro Biochemical Study of CYP51-Mediated Azole Resistance in Aspergillus fumigatus

Novel Substrate Specificity and Temperature-Sensitive Activity of Mycosphaerella graminicola CYP51 Supported by the Native NADPH Cytochrome P450 Reductase

Heterologous expression and characterization of the sterol 14α-demethylase CYP51F1 from Candida albicans

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