Search Results (19)

The increased activity of δ-aminolevulinic acid synthase-1 (ALAS1) leads to the accumulation of δ-aminolevulinic acid (ALA), which may reduce the iron(III) retention ability of ferritin, resulting in iron(III) overload. The accumulation of ALA occurs in the condition of attenuation of the heme negative feedback loop over ALAS1 activity in concert with the induction of ALAS1 expression. Attenuation of the heme negative feedback loop is maintained by elevated quantities of the heme catabolizing enzyme, heme oxygenase-1 (HO-1), which, in turn, may be induced by highly increased heme concentration. The upregulation of brain HO-1 occurs in patients with Alzheimer’s and Parkinson’s diseases. A mouse with overexpressed human HMOX1 is a model of schizophrenia with concurrent iron overload. HO-1 inhibitors reduce oxidative damage to whole cells and mitochondrial compartments of rat astrocytes transfected with the HMOX1 gene. Iron reduction within the heme prosthetic moiety of P450 cytochromes in microsomes is facilitated by NADPH-cytochrome P450 oxidoreductase (CPR), while adrenodoxin reductase performs this function in the mitochondria. In partial CPR-deficient conditions, the half-life of apocytochromes is prolonged while HO-1 is induced due to the elevated heme release from unreduced cytochromes. This study posits that, before being degraded by HO-1, the released hemin triggers the oligomerization of cytochromes, as well as other oxidative damages, by producing hydroperoxyl radicals from hydrogen peroxide produced in uncoupling reaction. After reaching a specific level, iron(III) overload may trigger the saturation of CPR, resulting in the development of neuropathologies. Full article

NADPH–cytochrome P450 reductase (CPR) is an essential redox partner for a wide range of metal-containing proteins, mediating the stepwise transfer of two electrons from nicotinamide adenine dinucleotide phosphate (NADPH) to the redox centers of its partner proteins. This Perspective summarizes recent advances in understanding the mechanisms underlying the CPR-mediated electron transfer (ET) cycle. Emphasis is placed on human and other mammalian CPRs, which provide critical insights into human biology and drug metabolism. Recent experimental and computational approaches that have deepened our mechanistic understanding of CPR function are highlighted. Selected studies are reviewed to illustrate progress in elucidating the interflavin ET within CPR, the interplay between its redox states and structural dynamics, and its protein–protein interactions with redox partners, along with the associated ET pathways. Finally, the remaining challenges and future research directions are outlined. Full article

The activity of cytochrome P450 enzymes decreases in older adults, which can lead to toxic effects from polypharmacy. Cytochromes P450 are the most significant enzymes involved in the metabolism of foreign compounds, including pharmaceutical substances. Vitamin B2, or riboflavin (RF), is a potent antioxidant that is vital for the body and participates in numerous enzyme-catalyzed redox reactions. RF is phosphorylated intracellularly to form flavin mononucleotide (FMN), which is further metabolized into flavin adenine dinucleotide (FAD). The active site of the NADPH-dependent cytochrome P450 reductase (CPR), a redox partner of CYP enzymes, is necessary for the catalytic functions of cytochromes P450. The active site of reductase is a complex formed by two types of vitamin B2, such as flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN). In our study, we investigated the impact of the phosphorylated form of vitamin B2, FAD, and FMN on the catalytic activity of cytochrome P450 2C9 (CYP2C9) towards non-steroidal anti-inflammatory medications diclofenac and naproxen. It was shown that FAD significantly enhanced the catalytic efficiency of CYP2C9. The 4-hydroxylation of diclofenac was enhanced by 148 ± 10%. The O-demethylation of naproxen showed an increase of 120 ± 14%. Based on these data, we can assume that intake of vitamin B2 (riboflavin) improves catalytic efficiency of CYP2C9. This finding is essential for the modulation of catalytic activity of CYP2C9. The proposed electroanalytic approach is a sensitive and robust method for drug metabolism assay. Full article

The Colorado Potato Beetle (CPB, Leptinotarsa decemlineata Say, Coleoptera: Chrysomelidae) remains a destructive agricultural pest worldwide that continually overcomes conventional control methods. In recent years, RNA interference (RNAi) has emerged as an alternative for its management; however, although promising results have been reported, its effectiveness has been influenced by several factors, including the length of double-stranded RNA (dsRNA), the delivery method, stability, and especially the selection of the target gene. In this study, we designed and synthesized 290 bp dsRNAs targeting the SERCA and CPR genes from L. decemlineata, which encode the Sarco/Endoplasmic Reticulum Ca²⁺-ATPase and NADPH–Cytochrome P450 Reductase, respectively. Both dsRNAs successfully reduced transcript levels in larvae, with dsSERCA achieving ~60% knockdown by day 3 and dsCPR achieving ~50% knockdown by day 7. Furthermore, both treatments affected the larval growth and survival rate. However, while the dsCPR-treated larvae showed a 59% reduction in weight gain, the administration of dsSERCA had a strong phenotypic effect on the larvae, leading to decreased feeding, a 50.4% reduction in weight gain, and ultimately, 100% mortality. These results suggest that the SERCA and CPR genes could be promising targets for L. decemlineata control and emphasize the importance of appropriate target gene selection for RNAi silencing, as well as the need to explore and validate new genes for RNAi-mediated pest management. Full article

Mammalian heme oxygenase (HO) catalyzes heme degradation using reducing equivalents supplied by NADPH–cytochrome P450 reductase (CPR). The tertiary structure of the catalytic domain of a constitutively expressed isoform of HO, HO-2, resembles that of the inductive isoform, HO-1, whereas HO-2 has two heme regulatory motifs (HRM) at the proximal portion of the C-terminus, where the disulfide linkage reflects cellular redox conditions and the second heme binding site is located. Here, we report the results of crosslinking experiments, which suggest that HRM is located near the FMN-binding domain of the CPR when it is complexed with HO-2. The enzymatic assay and reduction kinetics results suggest that heme-bound HRM negatively regulates HO-2 activity in vitro. Cellular redox conditions and free heme concentrations may regulate HO-2 activity. Full article

The hydroxylation of steroids in the C7β position is one of the rare reactions that allow the production of value-added precursors in the synthesis of ursodeoxycholic acid and other pharmaceuticals. Recently, we discovered this activity in the ascomycete Curvularia sp. VKM F-3040. In this study, the novel gene of 7-hydroxylase (P450_cur) was identified as being heterologously expressed and functionally characterized in Pichia pastoris. Transcriptome data mining and differential expression analysis revealed that 12 putative genes in Curvularia sp. mycelia significantly increased their expression in response to dehydroepiandrosterone (DHEA). The transcriptional level of the most up-regulated cytochrome P450_cur gene was increased more than 300-fold. A two-gene construct with a candidate P450_cur gene and the gene of its natural redox partner, NADPH-cytochrome P450 reductase (CPR), which is interconnected by a T2A element, was created. Using this construct, recombinant P. pastoris strains co-expressing fungal P450_cur and CPR genes were obtained. The functional activity of the recombinant P450_cur was studied in vivo during the bioconversion of androstane steroids. The fungal 7-monooxygenase predominantly catalyzed the 7β-hydroxylation of androstadienedione (ADD), DHEA, and androstenediol, whereas 1-dehydrotestosterone was hydroxylated by P450_cur mainly at the C7-Hα position. To our knowledge, this is the first report of a recombinant yeast capable of catalyzing the 7α/β-hydroxylation of ADD and DHEA. Full article

Diterpenoid alkaloids (DAs) are major pharmacologically active ingredients of Aconitum vilmorinianum, an important medicinal plant. Cytochrome P450 monooxygenases (P450s) are involved in the DA biosynthetic pathway, and the electron transfer reaction of NADPH-cytochrome P450 reductase (CPR) with P450 is the rate-limiting step of the P450 redox reaction. Here, we identified and characterized two homologs of CPR from Aconitum vilmorinianum. The open reading frames of AvCPR1 and AvCPR2 were found to be 2103 and 2100 bp, encoding 700 and 699 amino acid residues, respectively. Phylogenetic analysis characterized both AvCPR1 and AvCPR2 as class II CPRs. Cytochrome c and ferricyanide could be reduced with the recombinant proteins of AvCPR1 and AvCPR2. Both AvCPR1 and AvCPR2 were expressed in the roots, stems, leaves, and flowers of A. vilmorinianum. The expression levels of AvCPR1 and AvCPR2 were significantly increased in response to methyl jasmonate (MeJA) treatment. The yeasts co-expressing AvCPR1/AvCPR2/SmCPR1 and CYP76AH1 all produced ferruginol, indicating that AvCPR1 and AvCPR2 can transfer electrons to CYP76AH1 in the same manner as SmCPR1. Docking analysis confirmed the experimentally deduced functional activities of AvCPR1 and AvCPR2 for FMN, FAD, and NADPH. The functional characterization of AvCPRs will be helpful in disclosing molecular mechanisms relating to the biosynthesis of diterpene alkaloids in A. vilmorinianum. Full article

Ginsenosides are major bioactive compounds found in Panax ginseng that exhibit various pharmaceutical properties. Dammarenediol-II, the nucleus of dammarane-type ginsenosides, is a promising candidate for pharmacologically active triterpenes. Dammarenediol-II synthase (DDS) cyclizes 2,3-oxidosqualene to produce dammarenediol-II. Based on the native terpenoids synthetic pathway, a dammarane-type ginsenosides synthetic pathway was established in Chlamydomonas reinhardtii by introducing P. ginseng PgDDS, CYP450 enzyme (PgCYP716A47), or/and Arabidopsis thaliana NADPH-cytochrome P450 reductase gene (AtCPR), which is responsible for producing dammarane-type ginsenosides. To enhance productivity, strategies such as “gene loading” and “culture optimizing” were employed. Multiple copies of transgene expression cassettes were introduced into the genome to increase the expression of the key rate-limiting enzyme gene, PgDDS, significantly improving the titer of dammarenediol-II to approximately 0.2 mg/L. Following the culture optimization in an opt2 medium supplemented with 1.5 mM methyl jasmonate under a light:dark regimen, the titer of dammarenediol-II increased more than 13-fold to approximately 2.6 mg/L. The C. reinhardtii strains engineered in this study constitute a good platform for the further production of ginsenosides in microalgae. Full article

The use of marine enzymes as catalysts for biotechnological applications is a topical subject. Marine enzymes usually display better operational properties than their animal, plant or bacterial counterparts, enlarging the range of possible biotechnological applications. Due to the fact that cytochrome P450 enzymes can degrade many different toxic environmental compounds, these enzymes have emerged as valuable tools in bioremediation processes. The present work describes the isolation, purification and biochemical characterization of a liver NADPH-dependent cytochrome P450 reductase (CPR) from the marine fish Liza klunzingeri (LkCPR). Experimental results revealed that LkCPR is a monomer of approximately 75 kDa that is active in a wide range of pH values (6–9) and temperatures (40–60 °C), showing the highest catalytic activity at pH 8 and 50 °C. The activation energy of the enzyme reaction was 16.3 kcal mol⁻¹ K⁻¹. The K_M values for cytochrome C and NADPH were 8.83 μM and 7.26 μM, and the k_cat values were 206.79 s⁻¹ and 202.93 s⁻¹, respectively. LkCPR displayed a specific activity versus cytochrome C of 402.07 µmol min⁻¹ mg¹, the highest activity value described for a CPR up to date (3.2–4.7 times higher than the most active reported CPRs) and showed the highest thermostability described for a CPR. Taking into account all these remarkable catalytic features, LkCPR offers great potential to be used as a suitable biocatalyst. Full article

Cryptococcus remains a leading cause of invasive fungal infections in immunocompromised people. Resistance to azole drugs has imposed a further challenge to the effective treatment of such infections. In this study, the functional expression of full-length hexahistidine-tagged Cryptococcus neoformans CYP51 (CnCYP51-6×His), with or without its cognate hexahistidine-tagged NADPH-cytochrome P450 reductase (CnCPR-6×His), in a Saccharomyces cerevisiae host system has been used to characterise these enzymes. The heterologous expression of CnCYP51-6×His complemented deletion of the host CYP51 and conferred increased susceptibility to both short-tailed and long-tailed azole drugs. In addition, co-expression of CnCPR-6×His decreased susceptibility 2- to 4-fold for short-tailed but not long-tailed azoles. Type 2 binding of azoles to CnCYP51-6×His and assay of NADPH cytochrome P450 reductase activity confirmed that the heterologously expressed CnCYP51 and CnCPR are functional. The constructs have potential as screening tools and use in structure-directed antifungal discovery. Full article

NADPH-cytochrome P450 reductase (CPR) from Sorghum bicolor (SbCPR) serves as an electron donor for cytochrome P450 essential for monolignol and lignin production in this biofuel crop. The CPR enzymes undergo an ample conformational transition between the closed and open states in their functioning. This transition is triggered by electron transfer between the FAD and FMN and provides access of the partner protein to the electron-donating FMN domain. To characterize the electron transfer mechanisms in the monolignol biosynthetic pathway better, we explore the conformational transitions in SbCPR with rapid scanning stop-flow and pressure-perturbation spectroscopy. We used FRET between a pair of donor and acceptor probes incorporated into the FAD and FMN domains of SbCPR, respectively, to characterize the equilibrium between the open and closed states and explore its modulation in connection with the redox state of the enzyme. We demonstrate that, although the closed conformation always predominates in the conformational landscape, the population of open state increases by order of magnitude upon the formation of the disemiquinone state. Our results are consistent with several open conformation sub-states differing in the volume change (ΔV⁰) of the opening transition. While the ΔV⁰ characteristic of the oxidized enzyme is as large as −88 mL/mol, the interaction of the enzyme with the nucleotide cofactor and the formation of the double-semiquinone state of CPR decrease this value to −34 and −18 mL/mol, respectively. This observation suggests that the interdomain electron transfer in CPR increases protein hydration, while promoting more open conformation. In addition to elucidating the functional choreography of plant CPRs, our study demonstrates the high exploratory potential of a combination of the pressure-perturbation approach with the FRET-based monitoring of protein conformational transitions. Full article

Nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase (CPR) is an essential enzyme that transfers electrons from NADPH to cytochrome P450 monooxygenases. CPR is involved in cuticular hydrocarbon (CHC) synthesis in insects and is vital for insect development and survival. Here, we clarify the physiological function of a CPR gene in Nilaparvata lugens, an important rice pest, by using RNA interference. CPR gene knockdown leads to the functional loss of waterproofing and water retention in the integument of female adults, which causes significantly reduced body weight and a lethal phenotype. Scanning electron microscopy shows that the lipid layer on the outermost surface of the abdominal cuticle becomes thin in dsCPR-injected adults. Furthermore, CHC profile analysis reveals that CPR knockdown significantly decreases the contents of CHCs with a carbon chain length ≥ C27 in adult females. Moreover, we find that CPR knockdown generates a deficient phenotype in ovaries with deformed oocytes and a complete failure of egg-laying. These findings suggest that CPR plays multiple functional roles in CHC biosynthesis and embryo development in insects. Full article

The fungal cytochrome P450 lanosterol 14α-demethylase (CYP51) is required for the biosynthesis of fungal-specific ergosterol and is the target of azole antifungal drugs. Despite proven success as a clinical target for azole antifungals, there is an urgent need to develop next-generation antifungals that target CYP51 to overcome the resistance of pathogenic fungi to existing azole drugs, toxic adverse reactions and drug interactions due to human drug-metabolizing CYPs. Candida parapsilosis is a readily transmitted opportunistic fungal pathogen that causes candidiasis in health care environments. In this study, we have characterised wild type C. parapsilosis CYP51 and its clinically significant, resistance-causing point mutation Y132F by expressing these enzymes in a Saccharomyces cerevisiae host system. In some cases, the enzymes were co-expressed with their cognate NADPH-cytochrome P450 reductase (CPR). Constitutive expression of CpCYP51 Y132F conferred a 10- to 12-fold resistance to fluconazole and voriconazole, reduced to ~6-fold resistance for the tetrazoles VT-1161 and VT-1129, but did not confer resistance to the long-tailed triazoles. Susceptibilities were unchanged in the case of CpCPR co-expression. Type II binding spectra showed tight triazole and tetrazole binding by affinity-purified recombinant CpCYP51. We report the X-ray crystal structure of ScCYP51 in complex with VT-1129 obtained at a resolution of 2.1 Å. Structural analysis of azole—enzyme interactions and functional studies of recombinant CYP51 from C. parapsilosis have improved understanding of their susceptibility to azole drugs and will help advance structure-directed antifungal discovery. Full article

Ginseng (Panax ginseng C.A. Mey.) is a precious Chinese traditional medicine, for which ginsenosides are the most important medicinal ingredients. Cytochrome P450 enzymes (CYP450) and their primary redox molecular companion NADPH cytochrome P450 reductase (CPR) play a key role in ginsenoside biosynthesis pathway. However, systematic studies of CPR genes in ginseng have not been reported. Numerous studies on ginsenoside synthesis biology still use Arabidopsis CPR (AtCPR1) as a reductase. In this study, we isolated two CPR genes (PgCPR1, PgCPR2) from ginseng adventitious roots. Phylogenetic tree analysis showed that both PgCPR1 and PgCPR2 are grouped in classⅡ of dicotyledonous CPR. Enzyme experiments showed that recombinant proteins PgCPR1, PgCPR2 and AtCPR1 can reduce cytochrome c and ferricyanide with NADPH as the electron donor, and PgCPR1 had the highest enzymatic activities. Quantitative real-time PCR analysis showed that PgCPR1 and PgCPR2 transcripts were detected in all examined tissues of Panax ginseng and both showed higher expression in stem and main root. Expression levels of the PgCPR1 and PgCPR2s were both induced after a methyl jasmonate (MeJA) treatment and its pattern matched with ginsenoside accumulation. The present investigation suggested PgCPR1 and PgCPR2 are associated with the biosynthesis of ginsenoside. This report will assist in future CPR family studies and ultimately improving ginsenoside production through transgenic engineering and synthetic biology. Full article

The activity of microsomal cytochromes P450 (CYP) is strictly dependent on the supply of electrons provided by NADPH cytochrome P450 oxidoreductase (CPR). The variant nature of the isoform-specific proximal interface of microsomal CYPs implies that the interacting interface between the two proteins is degenerated. Recently, we demonstrated that specific CPR mutations in the FMN-domain (FD) may induce a gain in activity for a specific CYP isoform. In the current report, we confirm the CYP isoform dependence of CPR’s degenerated binding by demonstrating that the effect of four of the formerly studied FD mutants are indeed exclusive of a specific CYP isoform, as verified by cytochrome c inhibition studies. Moreover, the nature of CYP’s substrate seems to have a modulating role in the CPR:CYP interaction. In silico molecular dynamics simulations of the FD evidence that mutations induces very subtle structural alterations, influencing the characteristics of residues formerly implicated in the CPR:CYP interaction or in positioning of the FMN moiety. CPR seems therefore to be able to form effective interaction complexes with its structural diverse partners via a combination of specific structural features of the FD, which are functional in a CYP isoform dependent manner, and dependent on the substrate bound. Full article