Fatty acid dioxygenase-cytochrome P450 fusion enzymes of filamentous fungal pathogens

5,6-trans-AA (5,6-TAA, where TAA stands for trans-arachidonic acid) is a recently identified trans fatty acid that originates from the cis–trans isomerization of AA initiated by the NO2 radical. This trans fatty acid has been detected in blood circulation and we suggested that it functions as a lipid mediator of the toxic effects of NO2. To understand its role as a lipid mediator, we studied the metabolism of 5,6-TAA by liver microsomes stimulated with NADPH. Profiling of metabolites by liquid chromatography/MS revealed a complex mixture of oxidized products among which were four epoxides, their respective hydrolysis products (dihydroxyeicosatrienoic acids), and several HETEs (hydroxyeicosatetraenoic acids) resulting from allylic, bis-allylic and (ω−1)/(ω−2) hydroxylations. We found that the C5–C6 trans bond competed with the three cis bonds for oxidative metabolism mediated by CYP (cytochrome P450) epoxygenase and hydroxylase. This was evidenced by the detection of 5,6-trans-EET (where EET stands for epoxyeicosatrienoic acid), 5,6-erythro-dihydroxyeicosatrienoic acid and an isomer of 5-HETE. A standard of 5,6-trans-EET obtained by iodolactonization of 5,6-TAA was used for the unequivocal identification of the unique microsomal epoxide in which the oxirane ring was of trans configuration. Additional lipid products originated from the metabolism involving the cis bonds and thus these metabolites had the trans C5–C6 bond. The 5,6-trans-isomers of 18- and 19-HETE were likely to be products of the CYP2E1, because a neutralizing antibody partially inhibited their formation without having an effect on the formation of the epoxides. Our study revealed a novel pathway of microsomal oxidative metabolism of a trans fatty acid in which both cis and trans bonds participated. Of particular significance is the detection of the trans-epoxide of AA, which may be involved in the metabolic activation of such trans fatty acids and probably contribute to their biological activity. Unlike its cis-isomer, 5,6-trans-EET was significantly more stable and resisted microsomal hydrolysis and conjugation with glutathione catalysed by hepatic glutathione S-transferase.

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Binding of Cytochrome P450 Monooxygenase and Lipoxygenase Pathway Products by Heart Fatty Acid-Binding Protein†

Biochemistry ◽

10.1021/bi001602y ◽

2001 ◽

Vol 40 (4) ◽

pp. 1070-1076 ◽

Cited By ~ 55

Author(s):

Richard L. Widstrom ◽

Andrew W. Norris ◽

Arthur A. Spector

Keyword(s):

Cytochrome P450 ◽

Fatty Acid ◽

Fatty Acid Binding Protein ◽

Binding Protein ◽

Cytochrome P450 Monooxygenase ◽

Lipoxygenase Pathway ◽

P450 Monooxygenase ◽

Fatty Acid Binding ◽

Acid Binding Protein

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TheAspergillus fumigatusDamage Resistance Protein Family Coordinately Regulates Ergosterol Biosynthesis and Azole Susceptibility

mBio ◽

10.1128/mbio.01919-15 ◽

2016 ◽

Vol 7 (1) ◽

Cited By ~ 25

Author(s):

Jinxing Song ◽

Pengfei Zhai ◽

Yuanwei Zhang ◽

Caiyun Zhang ◽

Hong Sang ◽

...

Keyword(s):

Cytochrome P450 ◽

Fungal Pathogens ◽

Regulatory System ◽

Ergosterol Biosynthesis ◽

Heme Binding ◽

Cytochrome P450 Enzymes ◽

Azole Antifungals ◽

Ergosterol Synthesis ◽

Resistance Protein ◽

Cytochrome P450 Protein

ABSTRACTErgosterol is a major and specific component of the fungal plasma membrane, and thus, the cytochrome P450 enzymes (Erg proteins) that catalyze ergosterol synthesis have been selected as valuable targets of azole antifungals. However, the opportunistic pathogenAspergillus fumigatushas developed worldwide resistance to azoles largely through mutations in the cytochrome P450 enzyme Cyp51 (Erg11). In this study, we demonstrate that a cytochromeb5-like heme-binding damage resistance protein (Dap) family, comprised of DapA, DapB, and DapC, coordinately regulates the functionality of cytochrome P450 enzymes Erg5 and Erg11 and oppositely affects susceptibility to azoles. The expression of all three genes is induced in an azole concentration-dependent way, and the decreased susceptibility to azoles requires DapA stabilization of cytochrome P450 protein activity. In contrast, overexpression of DapB and DapC causes dysfunction of Erg5 and Erg11, resulting in abnormal accumulation of sterol intermediates and further accentuating the sensitivity of ΔdapAstrains to azoles. The results of exogenous-hemin rescue and heme-binding-site mutagenesis experiments demonstrate that the heme binding of DapA contributes the decreased azole susceptibility, while DapB and -C are capable of reducing the activities of Erg5 and Erg11 through depletion of heme.In vivodata demonstrate that inactivated DapA combined with activated DapB yields anA. fumigatusmutant that is easily treatable with azoles in an immunocompromised mouse model of invasive pulmonary aspergillosis. Compared to the single Dap proteins found inSaccharomyces cerevisiaeandSchizosaccharomyces pombe, we suggest that this complex Dap family regulatory system emerged during the evolution of fungi as an adaptive means to regulate ergosterol synthesis in response to environmental stimuli.IMPORTANCEKnowledge of the ergosterol biosynthesis route in fungal pathogens is useful in the design of new antifungal drugs and could aid in the study of antifungal-drug resistance mechanisms. In this study, we demonstrate that three cytochromeb5-like Dap proteins coordinately regulate the azole resistance and ergosterol biosynthesis catalyzed by cytochrome P450 proteins. Our new insights into the Dap regulatory system in fungal pathogens may have broad therapeutic ramifications beyond their usefulness for classic azole antifungals. Moreover, our elucidation of the molecular mechanism of Dap regulation of cytochrome P450 protein functionality through heme-binding activity may extend beyond the Kingdom Fungi with applicability toward Dap protein regulation of mammalian sterol synthesis.

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