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Amaryllidaceae alkaloids (AAs), such as galanthamine and lycorine, are natural products of Lycoris radiata possessing various pharmacological activities including anti-acetylcholinesterase, anti-inflammatory, and antitumour activities. Elucidating the biosynthesis of these special AAs is crucial for understanding their production and potential modification for improved clinical application, of which cytochrome P450 enzymes catalyse the formation of key alkaloid skeletons and subsequent modification processes, with the NAPDH cytochrome P450 reductases (CPRs) serving as essential redox partners. This study identified three CPRs, LrCPR1, LrCPR2, and LrCPR3, encoding 700, 697 and 695 amino acids, respectively, which belong to Class II CPRs. The LrCPRs reduced cytochrome c and ferricyanide in an NADPH-dependent manner, and their activities all followed the typical Michaelis-Menten curve. In yeast, the co-expression of LrCPRs and CYP96T6 produced the galantamine-like alkaloid namely N-demethylnarwedine, suggesting that they support the catalytic activity of CYP96T6. Quantitative analysis of the transcriptional expression profiles showed that LrCPRs were expressed in all the examined tissues of L. radiata, and their gene expression patterns are consistent with other genes that may be involved in the biosynthetic pathway of AAs, including cinnamate 4-hydroxylase and phenylalanine ammonia-lyase. Our study firstly provides the functional characterization of LrCPRs in L. radiata, which will contribute to the discovery of biosynthetic pathways and heterologous production of AAs.Key message: • Three LrCPRs genes were identified and characterized, which were further co-expressed with CYP96T6 in yeast, producing N-demethylnarwedine significantly. [ABSTRACT FROM AUTHOR]

Abstract NADPH-cytochrome P450 reductase (CPR) is a key enzyme transferring electrons to cytochrome P450. In tomatoes (Solanum lycopersicum), two CPR genes, SlCPR1 and SlCPR2, were identified. In all the tested tomato tissues, SlCPR2 showed higher expression levels than SlCPR1. SlCPR2 expression increased significantly with jasmonic acid treatment. No significant changes were observed with salicylic acid or drought stress treatment. The cDNA of SlCPRs were expressed in Escherichia coli without any amino acid modification. And the heterologously expressed SlCPR enzymes were reacted with several protein and chemical substrates. SlCPR2 was more active than SlCPR1. Both SlCPR1 and SlCPR2 exhibited strong activity across a pH range of 6.0 to 9.0, with peak activity at pH 8.0. The study opens possibilities for CPR control, biocatalyst development, and exploring oxidase enzyme functions.

NADPH-cytochrome P450 reductase (CPR) is a key enzyme transferring electrons to cytochrome P450. In tomatoes (Solanum lycopersicum), two CPR genes, SlCPR1 and SlCPR2, were identified. In all the tested tomato tissues, SlCPR2 showed higher expression levels than SlCPR1. SlCPR2 expression increased significantly with jasmonic acid treatment. No significant changes were observed with salicylic acid or drought stress treatment. The cDNA of SlCPRs were expressed in Escherichia coli without any amino acid modification. And the heterologously expressed SlCPR enzymes were reacted with several protein and chemical substrates. SlCPR2 was more active than SlCPR1. Both SlCPR1 and SlCPR2 exhibited strong activity across a pH range of 6.0 to 9.0, with peak activity at pH 8.0. The study opens possibilities for CPR control, biocatalyst development, and exploring oxidase enzyme functions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Fatty acid desaturases play an important role in maintaining the appropriate structure and function of biological membranes. The biochemical characterization of integral membrane desaturases, particularly ω3 and ω6 desaturases, has been limited by technical difficulties relating to the acquisition of large quantities of purified proteins, and by the fact that functional activities of these proteins were only tested in an NADH-initiated reaction system. The main aim of this study was to reconstitute an NADPH-dependent reaction system in vitro and investigate the kinetic properties of Mortierella alpina ω3 and ω6 desaturases in this system. After expression and purification of the soluble catalytic domain of NADPH–cytochrome P450 reductase, the NADPH-dependent fatty acid desaturation was reconstituted for the first time in a system containing NADPH, NADPH–cytochrome P450 reductase, cytochrome b5, M. alpina ω3 and ω6 desaturase and detergent. In this system, the maximum activity of ω3 and ω6 desaturase was 213.4 ± 9.0 nmol min−1 mg−1 and 10.0 ± 0.5 nmol min−1 mg−1, respectively. The highest kcat/Km value of ω3 and ω6 desaturase was 0.41 µM−1 min−1 and 0.09 µM−1 min−1 when using linoleoyl CoA (18:2 ω6) and oleoyl CoA (18:1 ω9) as substrates, respectively. M. alpina ω3 and ω6 desaturases were capable of using NADPH as reductant when mediated by NADPH–cytochrome P450 reductase; although, their efficiency is distinguishable from NADH-dependent desaturation. These results provide insights into the mechanisms underlying ω3 and ω6 fatty acid desaturation and may facilitate the production of important fatty acids in M. alpina.

The tomato leafminer, Tuta absoluta (Meyrick), one of the most economically destructive pests of tomato, causes severe yields losses of tomato production globally. Rapid evolution of insecticide resistance requires the development of alternative control strategy for this pest. RNA interference (RNAi) represents a promising, innovative control strategy against key agricultural insect pests, which has recently been licensed for Colorado Potato Beetle control. Here two essential genes, voltage-gated sodium channel (Na v) and NADPH-cytochrome P450 reductase (CPR) were evaluated as targets for RNAi using an ex vivo tomato leaf delivery system. Developmental stage-dependent expression profiles showed TaNa v was most abundant in adult stages, whereas TaCPR was highly expressed in larval and adult stages. T. absoluta larvae feeding on tomato leaflets treated with dsRNA targeting TaNa v and TaCPR showed significant knockdown of gene expression, leading to reduction in adult emergence. Additionally, tomato leaves treated with dsRNA targeting these two genes were significantly less damaged by larval feeding and mining. Furthermore, bioassay with LC 30 doses of λ -cyholthin showed that silencing TaNa v and TaCPR increased T. absoluta mortality about 32.2 and 17.4% , respectively, thus indicating that RNAi targeting TaNa v and TaCPR could increase the susceptibility to λ -cyholthin in T. absoluta. This study demonstrates the potential of using RNAi targeting key genes, like TaNa v and TaCPR , as an alternative technology for the control of this most destructive tomato pests in the future. [Display omitted] • RNAi targeting TaNa v and TaCPR caused significant knockdown of gene expression in T. absoluta. • RNAi targeting TaNa v and TaCPR resulted in the failure of adult emergence of T. absoluta. • RNAi targeting TaNa v and T aCPR reduced tomato leaf damage caused by T. absoluta larvae herbivory. • RNAi targeting TaNa v and TaCPR increased the susceptibility of T. absoluta larvae to λ -cyholthin. [ABSTRACT FROM AUTHOR]

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.

NADPH-cytochrome P450 reductase (CPR) is crucial for the detoxification process catalysed by cytochrome P450, which targets various exogenous xenobiotics, as well as pesticides. In our research, we successfully obtained the complete cDNA sequence of Apolygus lucorum 's CPR ( AlCPR ) using reverse transcription PCR along with rapid amplification of cDNA ends technology. Bioinformatics analysis exhibited that the inferred amino acid sequence of AlCPR is characteristic of standard CPRs, featuring an N-terminal membrane anchor and three conserved FMN, FAD and NADP binding sites. Phylogenetic result revealed that AlCPR was positioned within the Hemiptera cluster, showing a close evolutionary relationship with the CPR of Cimex lectularius . The real-time quantitative PCR results demonstrated widespread expression of AlCPR across various life stages and tissues of A. lucorum , with the most prominent expression in adults and the abdominal region. Injecting double-stranded RNA of AlCPR only significantly increased the lambda-cyhalothrin susceptibility in lambda-cyhalothrin-resistant strain rather than the susceptible strain. These findings suggest a potential link between AlCPR and the P450-dependent defence mechanism against lambda-cyhalothrin in A. lucorum.

Nicotinamide adenine dinucleotide phosphate (NADPH)‐cytochrome P450 reductase (CPR) is involved in the metabolism of endogenous and exogenous substances, and detoxification of insecticides. RNA interference (RNAi) of CPR in certain insects causes developmental defects and enhanced susceptibility to insecticides. However, the CPR of Acyrthosiphon pisum has not been characterized, and its function is still not understood. In this study, we investigated the biochemical functions of A. pisum CPR (ApCPR). ApCPR was found to be transcribed in all developmental stages and was abundant in the embryo stage, and in the gut, head, and abdominal cuticle. After optimizing the dose and silencing duration of RNAi for downregulating ApCPR, we found that ApCPR suppression resulted in a significant decrease in the production of cuticular and internal hydrocarbon contents, and of cuticular waxy coatings. Deficiency in cuticular hydrocarbons (CHCs) decreased the survival rate of A. pisum under desiccation stress and increased its susceptibility to contact insecticides. Moreover, desiccation stress induced a significant increase in ApCPR mRNA levels. We further confirmed that ApCPR participates in CHC production. These results indicate that ApCPR modulates CHC production, desiccation tolerance, and insecticide susceptibility in A. pisum, and presents a novel target for pest control. [ABSTRACT FROM AUTHOR]

Abstract Background Most plant cytochrome P450 (P450) proteins need to be supplied with electrons from a redox partner, e.g. an NADPH-cytochrome P450 reductase (CPR), for the activation of oxygen molecules via heme. CPR is a flavoprotein with an N-terminal transmembrane domain, which transfers electrons from NADPH to the P450 via coenzymes flavin adenine dinucleotide and flavin mononucleotide. Results In this study, a novel CPR (SdCPR) was isolated from a tropical medicinal plant Scoparia dulcis L. The deduced amino acid of SdCPR showed high homology of > 76% with CPR from higher plants and belonged to the class II CPRs of dicots. Recombinant SdCPR protein reduced cytochrome c, ferricyanide (K3Fe(CN)6), and dichlorophenolindophenol in an NADPH-dependent manner. To elucidate the P450 monooxygenase activity of SdCPR, we isolated a cinnamic acid 4-hydroxylase (SdC4H, CYP73A111) gene from S. dulcis. Biochemical characterization of SdCPR/SdC4H demonstrated that SdCPR supports the oxidation step of SdC4H. Real-time qPCR results showed that expression levels of SdCPR and SdC4H were inducible by mechanical wounding treatment and phytohormone elicitation (methyl jasmonate, salicylic acid), which were consistent with the results of promotor analyses. Conclusions Our results showed that the SdCPR and SdC4H are related to defense reactions, including the biosynthesis of secondary metabolites.

Fusarium species exhibit significant intrinsic resistance to most antifungal agents and fungicides, resulting in high mortality rates among immunocompromised patients. Consequently, a thorough characterization of the antifungal resistance mechanism is required for effective treatments and for preventing fungal infections and reducing antifungal resistance. In this study, an isolate of Fusarium oxysporum (wild-type) with broadly resistant to commonly antifungal agents was used to generate 1,450 T-DNA random insertion mutants via Agrobacterium tumefaciens -mediated transformation. Antifungal susceptibility test results revealed one mutant with increased sensitivity to azoles. Compared with the resistant wild-type, the mutant exhibited low MICs to KTZ, ITC, VRC, POS, and PCZ (0.125, 1, 0.06, 0.5, and 0.125μg/ml, respectively). The T-DNA insertion site of this mutant was characterized as involving two adjacent genes, one encoding a hypothetical protein with unknown function and the other encoding the NADPH-cytochrome P450 reductase, referred as CPR1. To confirm the involvement of these genes in the altered azole susceptibility, the independent deletion mutants were generated and the Cpr1 deletion mutant displayed the same phenotypes as the T-DNA random mutant. The deletion of Cpr1 significantly decreased ergosterol levels. Additionally, the expression of the downstream Cyp51 gene was affected, which likely contributed to the observed increased susceptibility to azoles. These findings verified the association between Cpr1 and azole susceptibility in F. oxysporum. Furthermore, this gene may be targeted to improve antifungal treatments. [ABSTRACT FROM AUTHOR]

Fusarium species exhibit significant intrinsic resistance to most antifungal agents and fungicides, resulting in high mortality rates among immunocompromised patients. Consequently, a thorough characterization of the antifungal resistance mechanism is required for effective treatments and for preventing fungal infections and reducing antifungal resistance. In this study, an isolate of Fusarium oxysporum (wild-type) with broadly resistant to commonly antifungal agents was used to generate 1,450 T-DNA random insertion mutants via Agrobacterium tumefaciens-mediated transformation. Antifungal susceptibility test results revealed one mutant with increased sensitivity to azoles. Compared with the resistant wild-type, the mutant exhibited low MICs to KTZ, ITC, VRC, POS, and PCZ (0.125, 1, 0.06, 0.5, and 0.125μg/ml, respectively). The T-DNA insertion site of this mutant was characterized as involving two adjacent genes, one encoding a hypothetical protein with unknown function and the other encoding the NADPH-cytochrome P450 reductase, referred as CPR1. To confirm the involvement of these genes in the altered azole susceptibility, the independent deletion mutants were generated and the Cpr1 deletion mutant displayed the same phenotypes as the T-DNA random mutant. The deletion of Cpr1 significantly decreased ergosterol levels. Additionally, the expression of the downstream Cyp51 gene was affected, which likely contributed to the observed increased susceptibility to azoles. These findings verified the association between Cpr1 and azole susceptibility in F. oxysporum. Furthermore, this gene may be targeted to improve antifungal treatments.

BACKGROUND The Asian citrus psyllid Diaphorina citri has developed high levels of resistance to many insecticides, and understanding its resistance mechanism will aid in the chemical control of this species. Nicotinamide adenine dinucleotide phosphate (NADPH)‐cytochrome P450 reductase (CPR) is crucial in cytochrome P450 function, and in some insects CPR knockdown has increased their susceptibility to insecticides. However, the CPR from D. citri has not been characterized and its function is undescribed. RESULTS: The CPR gene of D. citri (DcCPR) was cloned and sequenced. The expression level of DcCPR, determined by reverse‐transcription quantitative polymerase chain reaction (RT‐qPCR) analysis, was highest in the midgut and in nymphs. After feeding on double‐stranded RNA for 72 h, the DcCPR messenger RNA level in D. citri adults decreased by 68.4%, and the susceptibility of D. citri to imidacloprid and thiamethoxam significantly increased. Meanwhile, after DcCPR silencing, the specific activities of DcCPR protein and P450s were significantly reduced by 41.6% and 44.7%, respectively. The subsequent western blot analysis and quantification of band intensity also showed that DcCPR content significantly decreased, consistent with the results of the specific activity test. In a eukaryotic expression assay, the viability of cells expressing DcCPR was significantly higher than the viability of cells expressing green fluorescent protein (GFP) when cells were exposed to imidacloprid or thiamethoxam. CONCLUSION: These results indicate that DcCPR contributes to D. citri susceptibility to imidacloprid and thiamethoxam. [ABSTRACT FROM AUTHOR]

Background: Most plant cytochrome P450 (P450) proteins need to be supplied with electrons from a redox partner, e.g. an NADPH-cytochrome P450 reductase (CPR), for the activation of oxygen molecules via heme. CPR is a flavoprotein with an N-terminal transmembrane domain, which transfers electrons from NADPH to the P450 via coenzymes flavin adenine dinucleotide and flavin mononucleotide. Results: In this study, a novel CPR (SdCPR) was isolated from a tropical medicinal plant Scoparia dulcis L. The deduced amino acid of SdCPR showed high homology of > 76% with CPR from higher plants and belonged to the class II CPRs of dicots. Recombinant SdCPR protein reduced cytochrome c, ferricyanide (K3Fe(CN)6), and dichlorophenolindophenol in an NADPH-dependent manner. To elucidate the P450 monooxygenase activity of SdCPR, we isolated a cinnamic acid 4-hydroxylase (SdC4H, CYP73A111) gene from S. dulcis. Biochemical characterization of SdCPR/SdC4H demonstrated that SdCPR supports the oxidation step of SdC4H. Real-time qPCR results showed that expression levels of SdCPR and SdC4H were inducible by mechanical wounding treatment and phytohormone elicitation (methyl jasmonate, salicylic acid), which were consistent with the results of promotor analyses. Conclusions: Our results showed that the SdCPR and SdC4H are related to defense reactions, including the biosynthesis of secondary metabolites. [ABSTRACT FROM AUTHOR]

NADPH-cytochrome P450 reductase (CPR) is essential for the detoxification of endogenous and exogenous substances mediated by cytochrome P450. While several insect CPRs have been found to be associated with insecticide resistance, the CPR of Aphis gossypii has not been characterized, and its functional role in insecticide resistance remains undefined. In this study, we cloned and characterized the full-length sequence of A. gossypii CPR (AgCPR). The deduced amino acid sequence of AgCPR contains all conserved domains of CPR, which shows high similarity to other insect CPRs and was clustered into a same branch of aphids according to phylogenetic analysis. The transcript of AgCPR was present in all developmental stages, with the highest expression in the adult stage. Furthermore, the expression of AgCPR could be induced by sulfoxaflor, a commonly used insecticide, in a time- and dose-dependent manner. Further silencing of AgCPR by feeding dsRNA significantly increased the susceptibility of A. gossypii to this insecticide. These findings suggest that AgCPR may play a significant role in the susceptibility of A. gossypii to sulfoxaflor and in the development of P450-mediated resistance to sulfoxaflor. [Display omitted] • The full-length cDNA of A. gossypii CPR (AgCPR) was cloned and characterized. • AgCPR can be detected in all developmental stages and induced by sulfoxaflor. • Silencing of AgCPR increased susceptibility of resistant aphids to sulfoxaflor. [ABSTRACT FROM AUTHOR]

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.

BACKGROUND: Human hepatocyte in vitro cell culture systems are important models for drug development and toxicology studies in the context of liver xenobiotic metabolism. Often, such culture systems are used to elucidate the biotransformation of xenobiotics or drugs and further investigate drug and drug metabolite effects on biological systems in terms of potential therapeutic benefit or toxicity. Human hepatocytes currently used for such in vitro studies are mostly primary cells or cell lines derived from liver cancers. Both approaches have limitations such as low proliferation capacity and progressive dedifferentiation found in primary cells or lack of liver functions in cell lines, which makes it difficult to reliably predict biotransformation of xenobiotics in patients. In order to overcome these limitations, HepaFH3 cells and Upcyte® hepatocytes representing primary-like hepatocytes of the first and second generation are increasingly used. Based on primary human hepatocyte cells transduced for stable expression of Upcyte® proliferation genes, they are mitotically active and exhibit liver functions over an extended period, making them comparable to primary human hepatocytes. These hepatocyte models show active liver metabolism such as urea and glycogen formation as well as biotransformation of xenobiotics. The latter is based on the expression, activity and inducibility of cytochrome P450 enzymes (CYP) as essential phase I reaction components. However, for further characterisation in terms of performance and existing limitations, additional studies are needed to elucidate the mechanisms involved in phase I reactions. One prerequisite is sufficient activity of microsomal NADPH-cytochrome P450 reductase (POR) functionally connected as electron donor to those CYP enzymes. OBJECTIVE: For Upcyte® hepatocytes and HepaFH3 cells, it is so far unknown to what extent POR is expressed, active, and may exert CYP-modulating effects. Here we studied POR expression and corresponding enzyme activity in human hepatoblastoma cell line HepG2 and compared this with HepaFH3 and Upcyte® hepatocytes representing proliferating primary-like hepatocytes. METHODS: POR expression of those hepatocyte models was determined at mRNA and protein level using qRT-PCR, Western Blot and immunofluorescence staining. Kinetic studies on POR activity in isolated microsomes were performed by a colorimetric method. RESULTS: The investigated hepatocyte models showed remarkable differences at the level of POR expression. Compared to primary-like hepatocytes, POR expression of HepG2 cells was 4-fold higher at mRNA and 2-fold higher at protein level. However, this higher expression did not correlate with corresponding enzyme activity levels in isolated microsomes, which were comparable between all cell systems tested. A tendency of higher POR activity in HepG2 cells compared to HepaFH3 (p = 0.0829) might be present. Compared to primary human hepatocyte microsomes, POR activity was considerably lower in all hepatocyte models. CONCLUSION: In summary, our study revealed that POR expression and activity were clearly detectable in all in vitro hepatocyte models with the highest POR expression in cancer cell line HepG2. However, POR activity was lower in tested hepatocyte models when compared to human primary hepatocyte microsomes. Whether this was caused by e.g. polymorphisms or metabolic differences of investigated hepatocyte models will be target for future studies. [ABSTRACT FROM AUTHOR]

Abstract Dioscorea zingiberensis is a perennial medicinal herb rich in a variety of pharmaceutical steroidal saponins. Squalene epoxidase (SE) is the key enzyme in the biosynthesis pathways of triterpenoids and sterols, and catalyzes the epoxidation of squalene in coordination with NADPH-cytochrome P450 reductase (CPR). In this study, we cloned DzSE and DzCPR gene sequences from D. zingiberensis leaves, encoding proteins with 514 and 692 amino acids, respectively. Recombinant proteins were successfully expressed in vitro , and enzymatic analysis indicated that, when SE and CPR were incubated with the substrates squalene and NADPH, 2,3-oxidosqualene was formed as the product. Subcellular localization revealed that both the DzSE and DzCPR proteins are localized to the endoplasmic reticulum. The changes in transcription of DzSE and DzCPR were similar in several tissues. DzSE expression was enhanced in a time-dependent manner after methyl jasmonate (MeJA) treatments, while DzCPR expression was not inducible. Highlights • Optimal conditions for expression of key enzymes in diosgenin synthesis in E. coli. • DzSE and DzCPR cooperate with each other to synthesis 2,3-oxidosqualene in vitro. • Endoplasmic reticulum serves as the active site for squalene oxidation. • Tissue expression showed the synthesis, transport and storage of 2,3-oxidosqualene. [ABSTRACT FROM AUTHOR]

Abstract Cytochrome P450 reductase (CPR) is the redox partner of various P450s involved in primary and secondary metabolism. Here, we identified and characterized two paralogs of cytochrome P450 reductase from Siraitia grosvenorii. There were two full-length CPR isoforms in the S. grosvenorii fruit transcriptome dataset. They had the same open reading frames of 2, 124 bp, encoding 707 amino acids. A phylogenetic analysis characterized both SgCPR1 and SgCPR2 as Class II dicotyledonous CPRs. The recombinant proteins SgCPR1 and SgCPR2 could reduce cytochrome c and ferricyanide in a NADPH-dependent manner. The SgCPR1 and SgCPR2 transcripts were detected in all examined tissues of S. grosvenorii , and in fresh fruit, they had expression patterns similar to several key enzymes that require CPR as a partner during their biosynthesis. The expression levels of the SgCPR s were induced after a methyl jasmonate treatment. The extracts from yeast co-expressing SgCPR1/SgCPR2 and the cytochrome P450 enzyme CYP76AH1 produced ferruginol, indicating the positive effects of SgCPR1/SgCPR2 on the CYP76AH1 activity. A docking analysis confirmed the experimentally deduced functional activities of SgCPR1 and SgCPR2 for NADPH, FAD and FMN. Thus, SgCRP1 and SgCPR2 are both likely to participate in secondary metabolism, especially mogroside biosynthesis in S. grosvenorii. [ABSTRACT FROM AUTHOR]

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.

Ophiocordyceps sinensis Berk. is a fungal parasite that parasitizes the larvae of Hepialidae and is used as a traditional Chinese medicine. However, it is not clear how O. sinensis infects its host. The encoding gene haplotype diversity and predicted function of the nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase (CPR) related to the fungal pathogenicity was analyzed for 219 individuals from 47 O. sinensis populations. Two NADPH CPR genes of O. sinensis were detected and their dominant haplotypes were widely distributed throughout the entire distribution range in Western China. Only 5.43% of all O. sinensis individuals possessed the specific private haplotypes of NADPH CPR-1 and CPR-2 genes. Bioinformatic analyses predicted that the phosphorylation sites, motifs, and domains of NADPH CPR of O. sinensis were different between those encoding by the dominant and private gene haplotypes. The one-to-one match fungus–host correspondence of the same individual suggested that the widely distributed O. sinensis with the dominant NADPH CPR gene haplotypes may strongly infect almost all host insects through a random infection by oral or respiratory pores. Conversely, O. sinensis with the specific private NADPH CPR gene haplotypes is likely to infect only a few corresponding host insects by breaching the cuticle, due to the changed NADPH CPR structure and function.

NADPH-cytochrome P450 reductase (CPR) plays an essential role in the cytochrome P450 enzyme system, which aids in the metabolism of endogenous and exogenous compounds including the detoxification of insecticides. In this study, the CPR transcript in Aphis (Toxoptera) citricidus (Kirkaldy) was cloned, and the deduced amino acid sequence contained an N-terminal membrane anchor, three conserved binding domains (flavin mononucleotide, flavin adeninedinucleotide, and nicotinamide adenine dinucleotide phosphate), a flavin adeninedinucleotide-binding motif, and catalytic residues. Based on phylogenetic analysis, AcCPR was grouped in the hemipteran branch. AcCPR was ubiquitously expressed at all developmental stages and was most abundant in the adults and least abundant in third instar nymphs. Compared with other tested tissues of adults, the expression level of AcCPR was significantly high in the gut. Feeding double-stranded RNA of AcCPR reduced the AcCPR mRNA level and the activity of AcCPR in aphids, and the treated insects exhibited higher susceptibility to abamectin than the control group. Furthermore, the heterologous overexpression of AcCPR in Sf9 cells resulted in a greater viability than control cells when treated with abamectin. All results demonstrated that AcCPR may contribute to the resistance of A.citricidus to abamectin, and CPR may be a potential target for novel insecticide design or a new factor in the development of insecticide resistance.

The cytochrome P450 mixed function oxidase enzymes play a major role in the metabolism of important endogenous substrates as well as in the biotransformation of xenobiotics. The liver P450 system is the most active in metabolism of exogenous substrates. This review briefly describes the liver P450 (CYP) mixed function oxidase system with respect to its enzymatic components and functions. Electron transfer by the NADPH-P450 oxidoreductase is required for reduction of the heme of P450, necessary for binding of molecular oxygen. Binding of substrates to P450 produce substrate binding spectra. The P450 catalytic cycle is complex and rate-limiting steps are not clear. Many types of chemical reactions can be catalyzed by P450 enzymes, making this family among the most diverse catalysts known. There are multiple forms of P450s arranged into families based on structural homology. The major drug metabolizing CYPs are discussed with respect to typical substrates, inducers and inhibitors and their polymorphic forms. The composition of CYPs in humans varies considerably among individuals because of sex and age differences, the influence of diet, liver disease, presence of potential inducers and/or inhibitors. Because of such factors and CYP polymorphisms, and overlapping drug specificity, there is a large variability in the content and composition of P450 enzymes among individuals. This can result in large variations in drug metabolism by humans and often can contribute to drug–drug interactions and adverse drug reactions. Because of many of the above factors, especially CYP polymorphisms, there has been much interest in personalized medicine especially with respect to which CYPs and which of their polymorphic forms are present in order to attempt to determine what drug therapy and what dosage would reflect the best therapeutic strategy in treating individual patients.

NADPH-cytochrome P450 reductase delivers electrons required by heme oxygenase, squalene monooxygenase, fatty acid desaturase, and 48 human cytochrome P450 enzymes. While conformational changes supporting reductase intramolecular electron transfer are well defined, intermolecular interactions with these targets are poorly understood, in part because of their transient association. Herein the reductase FMN domain responsible for interacting with targets was fused to the N-terminus of three drug-metabolizing and two steroidogenic cytochrome P450 enzymes to increase the probability of interaction. These artificial fusion enzymes were profiled for their ability to bind their respective substrates and inhibitors and to perform catalysis supported by cumene hydroperoxide. Comparisons with the isolated P450 enzymes revealed that even the oxidized FMN domain causes substantial and diverse effects on P450 function. The FMN domain could increase, decrease, or not affect total ligand binding and/or dissociation constants depending on both P450 enzyme and ligand. As examples, FMN domain fusion has no effect on inhibitor ketoconazole binding to CYP17A1 but substantially altered CYP21A2 binding of the same compound. FMN domain fusion to CYP21A2 resulted in differential effects dependent on whether the ligand was 17α-hydroxyprogesterone versus ketoconazole. Similar enzyme-specific effects were observed on steady-state kinetics. These observations are most consistent with FMN domain interacting with the proximal P450 surface to allosterically impact P450 ligand binding and metabolism separate from electron delivery. The variety of effects on different P450 enzymes and on the same P450 with different ligands suggests intricate and differential allosteric communication between the P450 active site and its proximal reductase-binding surface., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

NADPH-cytochrome P450 reductase (CPR) plays an essential role in the cytochrome P450 enzyme system, which aids in the metabolism of endogenous and exogenous compounds including the detoxification of insecticides. In this study, the CPR transcript in Aphis (Toxoptera) citricidus (Kirkaldy) was cloned, and the deduced amino acid sequence contained an N-terminal membrane anchor, three conserved binding domains (flavin mononucleotide, flavin adeninedinucleotide, and nicotinamide adenine dinucleotide phosphate), a flavin adeninedinucleotide-binding motif, and catalytic residues. Based on phylogenetic analysis, AcCPR was grouped in the hemipteran branch. AcCPR was ubiquitously expressed at all developmental stages and was most abundant in the adults and least abundant in third instar nymphs. Compared with other tested tissues of adults, the expression level of AcCPR was significantly high in the gut. Feeding double-stranded RNA of AcCPR reduced the AcCPR mRNA level and the activity of AcCPR in aphids, and the treated insects exhibited higher susceptibility to abamectin than the control group. Furthermore, the heterologous overexpression of AcCPR in Sf9 cells resulted in a greater viability than control cells when treated with abamectin. All results demonstrated that AcCPR may contribute to the resistance of A. citricidus to abamectin, and CPR may be a potential target for novel insecticide design or a new factor in the development of insecticide resistance. [ABSTRACT FROM AUTHOR]

1. Hepatic drug-metabolizing activity was investigated in vitro with liver microsomes prepared from rats suffering from hypoxemia with experimentally induced acute lung impairment (ALI). 2. Male Wistar rats received an intrabronchial administration of dilute hydrochloride solution for ALI induction. Pooled liver microsomes were prepared for the normal and ALI rats, and the hepatic drug metabolism mediated by cytochrome P450 (CYP) 3 A was examined in an incubation study with the microsomes. 3. The NADPH-dependent metabolism of midazolam significantly increases in ALI rats as compared with that in normal rats. Testosterone 6β-hydroxylation was also observed to significantly increase in ALI rats. 4. When the hepatic expression of CYP3A proteins was examined, the protein expression of CYP3A1 was shown to significantly increase and that of CYP3A2 remained unaltered in ALI rats. The hepatic expression of NADPH-cytochrome P450 reductase (POR), a protein mediating electron transfer in CYP-mediated drug metabolism, was also revealed to significantly increases in ALI rats. 5. With the findings regarding the midazolam elimination, the hepatic drug-metabolizing activity seems to increase in response to acute hypoxemia, partly due to an altered expression of the CYP3A enzymes, and an augmented electron transfer with an increased POR expression is probably involved in the increase. [ABSTRACT FROM AUTHOR]

Background NADPH-cytochrome P450 reductase (CPR) plays important roles in cytochrome P450-mediated metabolism of endogenous and exogenous compounds, and participates in cytochrome P450-related detoxification of insecticides. However, the CPR from Locusta migratoria has not been well characterized and its function is still undescribed. Results The full-length of CPR gene from Locusta migratoria ( LmCPR ) was cloned by RT-PCR based on transcriptome information. The membrane anchor region, and 3 conserved domains (FMN binding domain, connecting domain, FAD/NADPH binding domain) were analyzed by bioinformatics analysis. Phylogenetic analysis showed that LmCPR was grouped in the Orthoptera branch and was more closely related to the CPRs from hemimetabolous insects. The LmCPR gene was ubiquitously expressed at all developmental stages and was the most abundant in the fourth-instar nymphs and the lowest in the egg stage. Tissue-specific expression analysis showed that LmCPR was higher expressed in ovary, hindgut, and integument. The CPR activity was relatively higher in Malpighian tubules and integument. Silencing of LmCPR obviously reduced the enzymatic activity of LmCPR, and enhanced the susceptibility of Locusta migratoria to carbaryl. Conclusion These results suggest that LmCPR contributes to the susceptibility of L. migratoria to carbaryl and could be considered as a novel target for pest control. [ABSTRACT FROM AUTHOR]

Nicotinamide adenine dinucleotide phosphate (NADPH)–cytochrome P450 reductase (CPR), a crucial electron-transfer partner of P450 systems, is required for various biological reactions catalyzed by P450 monooxygenase. Our previous study indicated that enhanced P450 enzyme detoxification and CYP6ER1 overexpression contributed to sulfoxaflor resistance in Nilaparvata lugens. However, the association between CPR , sulfoxaflor resistance, and neonicotinoid cross-resistance in N. lugens remains unclear. In this study, the sulfoxaflor-resistant (SFX-SEL) (RR = 254.04-fold), resistance-decline (DESEL) (RR = 18.99-fold), and susceptible unselected (UNSEL) strains of N. lugens with the same genetic background were established. Real-time quantitative polymerase chain reaction (RT-qPCR) revealed that the N. lugens CPR (NlCPR) expression level in the SFX-SEL strain was 6.85-fold and 6.07-fold higher than in UNSEL and DESEL strains, respectively. NlCPR expression was significantly higher in the abdomens of UNSEL, DESEL, and SFX-SEL fourth-instar nymphs than in other tissues (thoraxes, heads, and legs). Additionally, sulfoxaflor stress significantly increased NlCPR mRNA levels in the UNSEL, SFX-SEL and DESEL strains. NlCPR silencing by RNA interference (RNAi) dramatically increased the susceptibility of the UNSEL, DESEL, and SFX-SEL strains to sulfoxaflor, but the recovery of SFX-SEL was more obvious. Furthermore, NlCPR silencing led to a significant recovery in susceptibility to nitenpyram, dinotefuran, clothianidin, and thiamethoxam across all strains (UNSEL, DESEL, and SFX-SEL), with the greatest degree of recovery in the sulfoxaflor-resistant strain (SFX-SEL). Our findings suggest that NlCPR overexpression contributes to sulfoxaflor resistance and neonicotinoid cross-resistance in N. lugens. This will aid in elucidating the significance of CPR in the evolution of P450-mediated metabolic resistance in N. lugens. [Display omitted] • The expression level of NlCPR in the SFX-SEL strain was significantly upregulated compared with UNSEL and DESEL strain. • Sulfoxaflor stress significantly increased NlCPR mRNA levels in the UNSEL, DESEL, and SFX-SEL strains. • NlCPR silencing by RNAi dramatically increased the susceptibility of the SFX-SEL strain to sulfoxaflor. • NlCPR silencing significantly decreased the cross-resistance levels of SFX-SEL to four neonicotinoids. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a chronic neurodegenerative disease with an increasing morbidity rate. As one of the most important signaling pathways that responds to inflammation and degeneration, the p38 mitogen-activated protein kinase (MAPK) signaling pathway is active in the cortexes of AD mice. At the cellular level the same effect can be observed with p38 MAPK when induced by amyloid β (Aβ)1-42, a 42-residue Aβ fragment. Inhibition of p38 MAPK in the present study protected SH-SY5Y cells from the toxicity of Aβ1-42, and alleviated the formation of senile plaques and cognitive impairment in AD mice. The expression of cytochrome P450 reductase (CPR) in the brains of mice with AD, in addition to Aβ1-42-treated SH-SY5Y cells, also increased. However, the inhibition of CPR did not protect SH-SY5Y cells from the toxicity of Aβ1-42. The results of the present study suggest that p38 MAPK is a potential therapeutic target for the treatment of AD. In addition, the main enzyme that metabolizes drugs, CPR, could serve a more complex role in AD. [ABSTRACT FROM AUTHOR]

Precocene I is a juvenile hormone antagonist that needs to be activated via oxidative biotransformation catalyzed by cytochrome P450 (CYP). NADPH–cytochrome P450 reductase (CPR) supplies CYP with electrons in the oxidation–reduction process; however, its functional role in the activation of precocene I remains unexplored. Here, the representative characteristics of CPRs were analyzed in the CPR gene of Locusta migratoria (LmCPR), the result of model docking indicated that the hydrogen bonds were formed between reduced nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) and NADPH-, FAD-, FMN-domains of LmCPR, respectively. Treating the fourth-instar nymphs with precocene I decreased the juvenile hormone titers of nymphs to 0.55-fold of that in acetone-treated controls, and extended the interval time between fourth- and fifth-instar nymphs. 68.75% of the treated fourth-instar nymphs developed into precocious adults in the fifth-instar. LmCPR knockdown decreased the response to precocene I in the nymphs, the occurrence rate of precocious adults induced by precocene I treatment reduced by 23.11%. Therefore, LmCPR may be involved in the activation of precocene I in L. migratoria. In addition, we generated an active recombinant LmCPR protein using a prokaryotic expression system, its activity in reducing cytochrome c was 33.13 ± 11.50 nmol CytCred/min/μg protein. This study lays the foundation for further research on the role of LmCPR in precocene I activation. Figure. Model illustrating the involvement of LmCPR in the activation of prececone I. [Display omitted] • Precocene I treatment induced the occurrence of precocious adults. • LmCPR knockdown decreased the response to precocene I. • A recombinant LmCPR protein was successfully obtained by prokaryotic expression. [ABSTRACT FROM AUTHOR]

Objectives: To achieve heterologous biosynthesis of dammarenediol-II, which is the precursor of dammarane-type tetracyclic ginsenosides, by reconstituting the 2,3-oxidosqualene-derived triterpenoid biosynthetic pathway in Escherichia coli. Results: By the strategy of synthetic biology, dammarenediol-II biosynthetic pathway was reconstituted in E. coli by co-expression of squalene synthase (SS), squalene epoxidase (SE), NADPH-cytochrome P450 reductase (CPR) from Saccharomyces cerevisiae, and SE from Methylococcus capsulatus (McSE), NADPH-cytochrome P450 reductase (CPR) from Arabidopsis thaliana. Sequences of transmembrane domains were truncated if necessary in each of the genes. Different sources of SE/CPR combinations were tested, during which two CPRs were detected to be new reductase partners of McSE. When the gene encoding dammarenediol-II synthase was co-expressed with the 2,3-oxidosqualene expression modules, dammarenediol-II was detected and the production was 8.63 mg l in E. coli under the shake-flask conditions. Conclusions: Two E. coli chassis for production of dammarenediol-II were established which could be potentially applied in other triterpenoid production in E. coli when different oxidosqualene cyclases (OSCs) introduced into the system. [ABSTRACT FROM AUTHOR]

NADPH-cytochrome P450 reductase (CPR) plays an important role in cytochrome P450 function, and CPR knockdown in several insects leads to increased susceptibility to insecticides. However, a putative CPR gene has not yet been fully characterized in the small brown planthopper Laodelphax striatellus , a notorious agricultural pest in rice that causes serious damage by transmitting rice stripe and rice black-streaked dwarf viruses. The objective of this study was to clone the cDNA and to knock down the expression of the gene that encodes L. striatellus CPR ( LsCPR ) to further determine whether P450s are involved in the resistance of L. striatellus to buprofezin. First, the full-length cDNA of LsCPR was cloned and found to contain an open reading frame (ORF) encoding a polypeptide of 679 amino acids with a calculated molecular mass and isoelectric point of 76.92 kDa and 5.37, respectively. The deduced amino acid sequence shares high identity with the CPRs of other insects (98%, 97%, 75% and 68% for Sogatella furcifera , Nilaparvata lugens , Cimex lectularius and Anopheles gambiae , respectively) and possesses the characteristic features of classical CPRs, such as an N-terminal membrane anchor and conserved domains for flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) binding. Phylogenetic analysis revealed that LsCPR is located in a branch along with the CPRs of other hemipteran insects. LsCPR mRNA was detectable in all examined body parts and developmental stages of L. striatellus , as determined by real-time quantitative PCR (qPCR), and transcripts were most abundant in the adult abdomen and in first-instar nymphs and adults. Ingestion of 200 μg/mL of LsCPR double-stranded RNA (ds LsCPR ) by the planthopper for 5 days significantly reduced the transcription level of LsCPR . Moreover, silencing of LsCPR caused increased susceptibility to buprofezin in a buprofezin-resistant (YN-BPF) strain but not in a susceptible (YN) strain. These data further suggested that the P450-mediated metabolic detoxification of xenobiotics might be an important mechanism for buprofezin resistance in L. striatellus . [ABSTRACT FROM AUTHOR]

While the biodegradation of 2,4,6-trinitrotoluene (TNT) via the release of nitrite is well established, mechanistic details of the reaction in mammals are unknown. To address this issue, we attempted to identify the enzyme from rat liver responsible for the production of nitrite from TNT. A NADPH-cytochrome P450 reductase (P450R) was isolated and identified from rat liver microsomes as the enzyme responsible for not only the release of nitrite from TNT but also formation of superoxide and 4-hydroxyamino-2,6-dinitrotoluene (4-HADNT) under aerobic conditions. In this context, reactive oxygen species generated during P450R-catalyzed TNT reduction were found to be, at least in part, a mediator for the production of 4-HADNT from TNT via formation of 4-nitroso-2,6-dinitrotoluene. P450R did not catalyze the formation of the hydride-Meisenheimer complex (H - -TNT) that is thought to be an intermediate for nitrite release from TNT. Furthermore, in a time-course experiment, 4-HADNT formation reached a plateau level and then declined during the reaction between TNT and P450R with NADPH, while the release of nitrite was subjected to a lag period. Notably, the produced 4-HADNT can react with the parent compound TNT to produce nitrite and dimerized products via formation of a Janovsky complex. Our results demonstrate for the first time that P450R-mediated release of nitrite from TNT results from the process of chemical interaction of TNT and its 4-electron reduction metabolite 4-HADNT. [ABSTRACT FROM AUTHOR]

Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), tomato leaf miner, is one of the most devastating insect pests that attack tomato crops. T. absoluta larvae destroy tomato plants by producing large galleries in tomato leaves and burrowing stalks, apical buds, green and ripe fruits. This larva causes a major economic effect on tomato industry. Insecticides are commonly used to control this pest in tomato fields. The aim of this study is to determine the susceptibility levels of T. absoluta populations and biochemical analysis of mechanism(s) involved in chlorantraniliprole and metaflumizone metabolism, registered in Turkey for use against T. absoluta. IRAC bioassay method was used to determine the resistance ratios of these insecticides. Resistance ratios ranged between 7.2-and 1.7-folds for chlorantraniliprole, and 4.0- and 1.8-folds for metaflumizone in T. absoluta field populations. NADPH-Cytochrome P450 reductase, glutathione S-transferase, and esterase activities were determined by conducting biochemical assays in field populations of T. absoluta. While NADPH Cytochrome P450 reductase activity showed an increase only in Antalya population (1.9-fold), glutathione S-transferase activity showed an increase only in Adana population (1.2-fold). However, esterase activities were increased both in Adana (1.3-fold) and Ankara (2.2-fold) populations of T. absoluta. [ABSTRACT FROM AUTHOR]

Recently, it was found that the carbonyl group of 1-[3-(4-phenoxyphenoxy)-2-oxopropyl]indole-5-carboxylic acid ( 5), an inhibitor of the pro-inflammatory enzyme cytosolic phospholipase A2α, is easily reduced by rat liver S9 fractions in vitro. Determination of the inhibitory potency of certain putative inhibitors of carbonyl reducing enzymes on the transformation of the ketone derivative 5 to its alcohol 6 by recombinant microsomal NADPH-cytochrome P450 reductase and by recombinant cytosolic carbonyl reductase-1 now reveals that these compounds show a lack of specificity for these two enzymes in part. Thus, an assignment of the roles of different carbonyl reductases in metabolic keto reduction by the use of inhibitors is problematic. In addition, the ability of NADPH-cytochrome P450 reductase and carbonyl reductase-1 to reduce the ketone groups of the drugs haloperidol and daunorubicin was examined. Under the conditions applied, a pronounced reductive metabolism was only observed for daunorubicin in the presence of microsomal NADPH-cytochrome P450 reductase. Similarly, in rat liver S9 fractions a marked reduction of daunorubicin was seen, while haloperidol was only slightly metabolized to its alcohol. After separation of the S9 homogenate into a microsomal and a cytosolic fraction, it became evident that the ketone groups of daunorubicin, haloperidol and compound 5 were mainly reduced by cytosolic enzymes. However, since microsomes also catalysed these carbonyl reductions to some extent, it can be concluded that microsomal NADPH-cytochrome P450 reductase can contribute to metabolic keto reductions in xenobiotics. Copyright © 2015 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

NADPH-cytochrome P450 reductase was purified from Lake Van fish liver microsomes by primary and secondary DEAE-cellulose column chromatograph with 20.46 μM/min/mg enzyme specific activities, 54.4% purification yield, and purification of 38-fold. The purity of the enzyme was established, and its monomer molecular weight was determined by SDS-polyacrylamide gel electrophoresis. SDS-PAGE results showed a single band and the molecular weight of NADPH-cytochrome P450 reductase was 70 kDa. In addition, optimum ionic strength, optimum pH, optimum temperature, and stable pH values were determined for the enzyme in the kinetic studies performed. KM and Vmax were determined for NADPH and cytochrome c. Effects of some metals ions, antibiotics, and some other drugs used in aquarium fisheries on the activity of the enzyme were investigated. IC50 values and Ki values of metals showing an inhibitory effect were calculated. [ABSTRACT FROM AUTHOR]

BACKGROUND NADPH-cytochrome P450 reductase ( CPR) is essential for numerous biological reactions catalysed by microsomal cytochrome P450 monooxygenases (P450s). Knockdown of CPR in several insects leads to developmental defects and increased susceptibility to insecticides. However, information about the role of CPR in the brown planthopper, Nilaparvata lugens, is still unavailable. RESULTS A full-length cDNA encoding CPR was cloned from N. lugens ( NlCPR). The deduced amino acid sequence showed marked features of classical CPRs, such as an N-terminus membrane anchor, conserved domains for flavin mononucleotide, flavin adenine dinucleotide ( FAD) and nicotinamide adenine dinucleotide phosphate binding, as well as an FAD-binding motif and catalytic residues. Phylogenetic analysis revealed that NlCPR was located in a branch along with bed bug and pea aphid hemipteran insects. NlCPR mRNA was detectable in all tissues and developmental stages of N. lugens, as determined by real-time quantitative PCR. NlCPR transcripts were most abundant in the abdomen in adults, and in first-instar nymphs. Injection of N. lugens with double-strand RNA ( dsRNA) against NlCPR significantly reduced the transcription level of the mRNA, and silencing of NlCPR resulted in increased susceptibility in N. lugens to beta-cypermethrin and imidacloprid. CONCLUSION The results provide first evidence that NlCPR contributes to the susceptibility to beta-cypermethrin and imidacloprid in N. lugens. © 2014 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

The cDNAs for cytochrome P450 monooxygenase (designated as CYP97C27 by D. Nelson's group) and NADPH-cytochrome P450 reductase (designated as CPR I based on its classification) were isolated from Croton stellatopilosus leaves, which actively biosynthesise plaunotol (18-OH geranylgeraniol). CYP97C27 and CPR I contain open reading frames encoding proteins of 471 and 711 amino acids with predicted molecular masses of 53 and 79 kDa, respectively. By aligning the deduced sequences of CYP97C27 and CPR I with other plant species, all functional domains of CYP97C27 (heme and oxygen binding) and CPR I (CYP- and FMN, FAD, and NADPH cofactor binding) were identified. Amino acid sequence comparison indicated that both CYP97C27 (85–93%) and CPR I (79–83%) share high sequence identities with homologous proteins in other plant species, suggesting that CYP97C27 belongs to the CYP97 C subfamily and that CPR I belongs to class I of the dicotyledonous CPR. Functional characterisation of both enzymes, produced in Escherichia coli (pET32a/BL21(DE3)) as recombinant proteins, showed that simultaneous incubation of CYP97C27 and CPR I with the substrate geranylgeraniol (GGOH) and coenzyme NADPH led to formation of the product plaunotol. In C. stellatopilosus , the levels of the CYP97C27 and CPR I transcripts were highly correlated with those of several mRNAs involved in the plaunotol biosynthetic pathway, suggesting that CYP97C27 and CPR I are the enzymes that catalyse the last hydroxylation step of the pathway. [ABSTRACT FROM AUTHOR]

Picrorhiza kurrooa synthesizes a large array of pharmacologically important monoterpenoid iridoid glycosides called picrosides. Although chemical profile and pharmacological activities of P. kurrooa have been extensively studied, limited attempts have been made to decipher the biosynthetic route and to identify the key regulatory genes involved in picroside biosynthesis. In the present study, NADPH-cytochrome P450 reductase, a key enzyme involved in electron transfer to cytochrome P450s was identified from P. kurrooa. The full length cDNA (2679 bp) contained an open reading frame of 2133 bp, corresponding to 710 amino acids. PkCPR was heterologously expressed in Escherichia coli and the kinetic parameters of the recombinant enzyme were determined. Specific activity, V and K of PkCPR were found to be 5.8 ± 0.05 μmol min mg, 8.1 ± 0.12 μmol min mg and 7.8 μM, respectively. PkCPR was found to be spatially regulated at transcript level, being maximally expressed in leaf tissues. Altitude was found to have a positive effect on the picroside concentration and the picroside content positively correlated with the PkCPR transcript levels in samples collected at varied altitudes. Further, transcript profiling under methyl jasmonate, salicylic acid, 2,4-dicholorophenoxy acetic acid and UV-B elicitations displayed differential transcriptional regulation of PkCPR that fully corroborated with the identified cis-elements within the PkCPR promoter. Expression of PkCPR was inducible by UV-B and phytohormone elicitation, indicating that the PkCPR is possibly related to defence reactions, including biosynthesis of secondary metabolites. Present study is so far the only report of identification and functional characterization of CPR ortholog from P. kurrooa. [ABSTRACT FROM AUTHOR]

Abstract: CYP3A4 is the most abundant cytochrome P450 in the human liver. The expression level of CYP3A4 when coexpressed with cytochrome b 5 (cyt b 5) in Escherichia coli was 20–60% higher than that when it was expressed alone over an extended period (48–72h). This time-dependent elevation in coexpression with cyt b 5 was a result of an increase in CYP3A4 mRNA half-life; no significant change in CYP3A4 degradation was seen in the bacterial protease fraction. These results suggest that the higher CYP3A4 levels observed upon coexpression with cyt b 5 primarily resulted from CYP3A4 mRNA stabilization by cyt b 5. [Copyright &y& Elsevier]

The aim of the present study was to test a new hypothesis that brain cytochrome P450 reductase (CPR) and CPR-dependent enzymes play important roles in behavioral performance. A mouse model with brain neuron-specific deletion of the Cpr gene (brain- Cpr -null) was recently generated. Brain- Cpr -null mice and wild-type (WT) littermates were compared in a variety of behavioral assays. Notable differences were found in the exploratory behavior assay: for both males and females, activity in the center of the chamber was significantly higher for brain- Cpr -null than for WT mice on days 2 and 3 of the assay, although no significant difference was found between the two groups in anxiety-like behavior in the elevated zero maze. Furthermore, in the fear-conditioning assay, brain- Cpr -null mice exhibited significantly less activity suppression than did WT controls. This deficit in activity suppression was not accompanied by any difference between WT and brain- Cpr -null mice in nociceptive responses to foot shocks. Abnormal activity suppression was also observed in both male and female brain- Cpr -null mice during the contextual memory test. However, in the Morris water maze assay, the brain- Cpr -null and WT mice were indistinguishable, indicating normal spatial memory in the mutant mice. These data collectively indicate a novel role of the Cpr gene in fear conditioning and memory. [ABSTRACT FROM AUTHOR]

Benzo(a)pyrene (B(a)P) is a well-known environmental contaminant and carcinogen. Its sources include tobacco smoke, automobile exhaust, forest fire, and other combustion processes. Farnesol, an active principle of Vachellia farnesiana and other aromatic plants, possesses preventive properties against various toxicities. Present study was designed to estimate chemopreventive effects of farnesol against B(a)P-induced pulmonary injuries. To determine the protective effects of farnesol, it was administered orally at 2 doses (100 and 200 mg/kg body weight [[b.w.]]) once daily for 14 days. Rats were exposed intratracheally to B(a)P, 5 mg/kg b.w. on days 12 and 14, thereafter assessed for pulmonary toxicities 24 hours post last dose of B(a)P. B(a)P-induced edema, inflammation, oxidative stress, and consequent damages in lungs were assessed in terms of total protein, total cell count, nitric oxide (NO), lactate dehydrogenase (LDH), alkaline phosphatase, and in bronchoalveolar lavage fluid (BALF). B(a)P also reduced the levels of phospholipids (lung surfactants) in BALF. However, pretreatment with farnesol at both the doses significantly reduced the lung injuries and inflammatory responses. Farnesol also protected the levels of phospholipids to normal when compared with control. It also modified the activities of B(a)P metabolizing enzymes NADPH-cytochrome P450 reductase, microsomal epoxide hydrolase (mEH), and glutathione S-transferase (GST) in lung tissue of rats. Present findings suggest a prominent role of farnesol against B(a)P-induced lung inflammation, edema, surfactant dysfunction, and epithelial damages in Wistar rats. In conclusion, farnesol shows lung protection against B(a)P toxicities in Wistar rats. [ABSTRACT FROM AUTHOR]

Abstract: 7-Dehydrocholesterol reductase (DHCR7) catalyzes the final step in cholesterol synthesis. The enzyme utilizes NADPH as a source of electrons and has been reported to require NADPH-cytochrome P450 reductase (POR) as its redox partner. To test this hypothesis, microsomes were prepared from the livers of mice in which hepatic cytochrome P450 reductase expression was extinguished during maturation. These microsomes contained negligible levels of POR but had 2.5-fold greater DHCR7 activity than did microsomes from wild-type mice. Consistent with this greater activity, immunoblot analysis of DHCR7 expression indicated that DHCR7 protein levels were elevated 2-fold in POR-null microsomes. Addition of POR to these microsomes provided no stimulation of DHCR7 activity, confirming the lack of a role for POR in DHCR7 activity. Because the original observation that POR was necessary for DHCR7 activity was based, in part, on antibody inhibition studies with POR antibody, the ability of an antibody to the full-length POR protein to inhibit DHCR7 activity and cytochrome c reductase activity was tested; the antibody had no effect on DHCR7 activity but decreased cytochrome c reductase activity (a POR-catalyzed reaction) by 50%. Immunoblot analysis further demonstrated no cross-reactivity between POR and DHCR7 with antibodies to either protein. We conclude that cytochrome P450 reductase is not involved in 7-dehydrocholesterol reductase activity. [Copyright &y& Elsevier]

Abstract: Heme oxygenase (HO) catalyses the degradation of heme to biliverdin, carbon monoxide (CO) and ferrous iron via three successive monooxygenase reactions, using electrons provided by NADPH-cytochrome P450 reductase (CPR) and oxygen molecules. For cleavage of the oxaporphyrin ring of ferrous α-verdoheme, an intermediate in the HO reaction, involvement of a verdoheme π-neutral radical has been proposed. To explore this hypothetical mechanism, we performed electrochemical reduction of ferrous α-verdoheme–rat HO-1 complex under anaerobic conditions. Upon binding of CO, an O2 surrogate, the midpoint potential for one-electron reduction of the oxaporphyrin ring of ferrous α-verdoheme was increased from −0.465 to −0.392V vs the normal hydrogen electrode. Because the latter potential is close to that of the semiquinone/reduced redox couple of FAD in CPR, the one-electron reduction of the oxaporphyrin ring of CO-bound verdoheme complexed with HO-1 is considered to be a thermodynamically likely process. Indeed the one-electron reduced species, [FeII(verdoheme•)], was observed spectroscopically in the presence of CO in both NADPH/wild-type and FMN-depleted CPR systems under anaerobic conditions. Under physiological conditions, therefore, it is possible that O2 initially binds to the ferrous iron of α-verdoheme in its complex with HO-1 and an electron is subsequently transferred from CPR, probably via FAD, to the oxaporphyrin ring. [ABSTRACT FROM AUTHOR]

Abstract: Objective: To search and analyze nitric oxide synthase (NOS) and similar proteins from Plasmodium berghei(Pb). Methods: The structure and function of nitric oxide synthase and similar proteins from Plasmodium berghei were analyzed and predicted by bioinformatics. Results: PbNOS were not available, but nicotinamide adenine dinucleotide 2′–phosphate reduced tetrasodium (NADPH)–cytochrome p450 reductase(CPR) were gained. PbCPR was in the nucleus of Plasmodium berghei, while 134aa–229aa domain was localize in nucleolar organizer. The amino acids sequence of PbCPR had the closest genetic relationship with Plasmodium vivax showing a 73% homology. The tertiary structure of PbCPR displayed the forcep–shape with wings, but no wings existed in the tertiary structure of its'' host, Mus musculus(Mm). 137aa–200aa, 201aa–218aa, 220aa–230aa, 232aa–248, 269aa–323aa, 478aa–501aa and 592aa–606aa domains of PbCPR showed no homology with MmCPRs'', and all domains were exposed on the surface of the protein. Conclusions: NOS can''t be found in Plasmodium berghei and other Plasmodium species. PbCPR may be a possible resistance site of antimalarial drug, and the targets of antimalarial drug and vaccine. It may be also one of the mechanisms of immune evasion. This study on Plasmodium berghei may be more suitable to Plasmodium vivax. And 137aa–200aa, 201aa–218aa, 220aa–230aa, 232aa–248, 269aa–323aa, 478aa–501aa and 592aa–606aa domains of PbCPR are more ideal targets of antimalarial drug and vaccine. [Copyright &y& Elsevier]

Abstract: The formation of reactive oxygen species by the cytochrome P450 monooxygenase system is thought to be due to autoxidation of NADPH-cytochrome P450 reductase and the nonproductive decay of oxygen-bound cytochrome P450 intermediates. To characterize this process in recombinant microsomal enzymes, we used a highly sensitive hydrogen peroxide assay based on Amplex red oxidation. This assay is 20 times more sensitive (LLD=5.0pmol/assay and LLQ=30pmol/assay) than the standard ferrous thiocyanate assay for detection of hydrogen peroxide. We found low, but detectable, spontaneous generation of hydrogen peroxide by recombinant human NADPH-cytochrome P450 reductase complexes (0.09nmol hydrogen peroxide/min/100Units of NADPH-cytochrome P450 reductase). Significantly higher rates of hydrogen peroxide production were observed when recombinant cytochrome P450 enzymes were coexpressed with NADPH-cytochrome P450 reductase (0.31nmol of hydrogen peroxide/min/100Units of NADPH-cytochrome P450 reductase). This was independent of the addition of any exogenous cytochrome P450 substrates. These data demonstrate that cytochrome P450s are a major source of hydrogen peroxide in the recombinant cytochrome P450 monooxygenase system. Moreover, substrate binding is not required for the cytochrome P450s to generate reactive oxygen species. [Copyright &y& Elsevier]

Abstract: Aromatase is an enzyme required for the conversion of androgens to estrogens. Estrogens are female sex hormones involved in the development and growth of breast tumors. It has been of significant interest to investigate the structure–function relationship of aromatase since its inhibitors have shown great promise in fighting breast cancer. Aromatase belongs to the cytochrome P450 family, and forms an electron-transfer complex with its partner, NADPH-cytochrome P450 reductase (CPR), during the aromatization reaction. Aromatase is found to be widely expressed in vertebrates with unique substrates androstenedione and testosterone, but with various catalytic capacities reflecting species differences in K m, V max, etc. This report will summarize current progress in sequence–function correlation analysis of the aromatase protein family and molecular characterization of the interaction between aromatase and CPR. These studies may lead to a novel field for the development of new inhibitors which interfere with the interaction between aromatase and CPR in order to inhibit the aromatization reaction. [Copyright &y& Elsevier]