Abstract
The eukaryotic microsomal cytochrome P450 systems consist of a cytochrome P450 enzyme (P450) and a cytochrome P450 redox partner, which generally is a cytochrome P450 reductase (CPR) that supplies electrons from NADPH. However, alternative electron donors may exist such as cytochrome b5 reductase and cytochrome b5 (CBR and CYB5, respectively) via, which is NADH-dependent and are also anchored to the endoplasmic reticulum. In the carotenogenic yeast Xanthophyllomyces dendrorhous, three P450-encoding genes have been described: crtS is involved in carotenogenesis and the CYP51 and CYP61 genes are both implicated in ergosterol biosynthesis. This yeast has a single CPR (encoded by the crtR gene), and a crtR - mutant does not produce astaxanthin. Considering that this mutant is viable, the existence of alternative cytochrome P450 electron donors like CBR and CYB5 could operate in this yeast. The aim of this work was to characterize the X. dendrorhous CBR encoding gene and to study its involvement in P450 reactions in ergosterol and carotenoid biosynthesis. Two CBRs genes were identified (CBR.1 and CBR.2), and deletion mutants were constructed. The two mutants and the wild-type strain showed similar sterol production, with ergosterol being the main sterol produced. The crtR - mutant strain produced a lower proportion of ergosterol than did the parental strain. These results indicate that even though one of the two CBR genes could be involved in ergosterol biosynthesis, crtR complements their absence in the cbr - mutant strains, at least for ergosterol production. The higher NADH-dependent cytochrome c reductase activity together with the higher transcript levels of CBR.1 and CYB5 in the crtR - mutant as well as the lower NADH-dependent activity in CBS-cbr.1- strongly suggest that CBR.1-CYB5 via participates as an alternative electron donor pathway for P450 enzymes involved in ergosterol biosynthesis in X. dendrorhous.
Highlights
The cytochrome P450s (P450s) constitute a large superfamily of heme-containing monooxygenases present in organisms from all domains of life [1,2]
The P450s act as a terminal electron acceptor in the multicomponent P450 dependent monooxygenation system (P450 systems) which leads to the reductive activation of molecular oxygen followed by the insertion of one oxygen atom into the substrate molecule, which catalyzes the following general reaction: RH + O2 + 2e− + 2H+ ! ROH + H2O, where R represents the substrate molecule [7]; the required electrons are generally supplied by NADPH and transferred to P450 by a P450 redox partner [8]
The results indicated that the alternative electron donor, probably CBR-CYB5, had a different affinity for the P450s enzymes CrtS, CYP51 and CYP61
Summary
The cytochrome P450s (P450s) constitute a large superfamily of heme-containing monooxygenases present in organisms from all domains of life [1,2] They play significant roles in the oxidative metabolism of a wide range of exogenous and endogenous substrates [3]. In the class II eukaryotic microsomal P450 systems, the general P450 redox partner is a cytochrome P450 reductase, termed CPR [1,7,9], and the electron flow goes from NADPH to FAD to FMN and to the heme group in P450. Alternative electron transfer mechanisms from NADH via cytochrome b5 reductase and cytochrome b5, which are anchored to the endoplasmic reticulum (CBR and CYB5), have been reported [10]. In cpr- mutant strains of the ascomycete Fusarium fujikuroi, the P450s activities involved in gibberellin biosynthesis showed changes in their regioselectivity, reaction rate and formation of alternative products due to their interaction with alternative redox partners [14]
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