NADPH-cytochrome P450 Reductase Research Articles

Multiple NADPH-cytochrome P450 reductases from Lycoris radiata involved in Amaryllidaceae alkaloids biosynthesis.

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.

Environmentally Persistent Free Radicals stimulate CYP2E1-mediated generation of reactive oxygen species at the expense of substrate metabolism.

Environmentally persistent free radicals (EPFRs) are a recently recognized component of particulate matter that cause respiratory and cardiovascular toxicity. The mechanism of EPFR toxicity appears to be related to their ability to generate reactive oxygen species (ROS), causing oxidative damage. EPFRs were shown to affect P450 function, inducing the expression of some forms through the Ah receptor. However, another characteristic of EPFRs lies in their ability to inhibit P450 activities. CYP2E1 is one of the P450s that is inhibited by EPFR (MCP230) exposure. As CYP2E1 is also known to generate ROS, it is important to understand the ability of EPFRs to influence the function of this enzyme and to identify the mechanisms involved. CYP2E1 was shown to be inhibited by EPFRs, and to a lesser extent by non-EPFR particles. As EPFR-mediated inhibition was more robust at subsaturating NADPH-cytochrome P450 reductase (POR) concentrations, disruption of POR·CYP2E1 complex formation and electron transfer were examined. Surprisingly, neither complex formation nor electron transfer between POR and CYP2E1 were inhibited by EPFRs. Examination of ROS production showed that MCP230 generated a greater amount of ROS than the non-EPFR CuO-Si. When a POR/CYP2E1-containing reconstituted system was added to the pollutant-particle systems there was a synergistic stimulation of ROS production. The results indicate that EPFRs cause inhibition of CYP2E1-mediated substrate metabolism, yet do not alter electron transfer and actually stimulate ROS generation. Taken together, the results are consistent with EPFRs affecting CYP2E1 function by inhibiting substrate metabolism and increasing the generation of ROS. Significance Statement Environmentally persistent free radicals affect CYP2E1 function by inhibition of monooxygenase activity. This inhibition is not due to disruption of the POR·CYP2E1 complex or inhibition of electron transfer, but due to uncoupling of NADPH and oxygen consumption from substrate metabolism to the generation of ROS. These results show that EPFRs block the metabolism of foreign compounds, and also synergistically stimulate the formation of reactive oxygen species that lead to oxidative damage within the organism.

NADPH-cytochrome P450 reductase involved in the lambda-cyhalothrin susceptibility on the green mirid bug Apolygus lucorum.

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.

Systematic metabolic engineering for improved synthesis of perillic acid in Candida tropicalis

Perillic acid has been studied as an anticancer and antimicrobial drug. Production of perillic acid has attracted considerable attention. Meanwhile, Candida tropicalis is an unconventional diploid yeast, most significantly characterized by its ability to metabolize alkanes or fatty acids for growth and proliferation. Therefore, perillic acid’s precursor (L-limonene) in C. tropicalis was firstly synthesized by expressing a Mentha spicata L-limonene synthase gene, LS_Ms in this work. Expression of a gene which encoded for a truncated version of tLS_Ms increased the production of L-limonene with a 2.78-fold increase in the titer over C. tropicalis GJR-LS-01. Compartmentalized expression of the gene tLS_Ms inhibited the production of L-limonene in C. tropicalis compared to cytoplasmic expression. Cytoplasmic overexpression of seven precursor synthesis genes significantly enhanced the production of L-limonene in C. tropicalis compared to their compartmentalized expression (mitochondria or peroxisomes), which increased by 31.7-fold in C. tropicalis GJR-tLS-01. The L-limonene titer in C. tropicalis GJR-EW-tLS-04 overexpressing the mutant gene ERG20WW in the cytoplasm was significantly increased, 11.33-fold higher than the control. The titer of L-limonene for 60 g/L glucose was increased by 1.40-fold compared to the control. Finally, a Salvia miltiorrhiza cytochrome P450 enzyme gene CYP7176 and an Arabidopsis thaliana NADPH cytochrome P450 reductase gene CPR were heterologously expressed in C. tropicalis GJR-EW-tLS-04C for the synthesis of perillic acid, which reached a titer of 106.69 mg/L in a 5-L fermenter. This is the first report of de novo synthesis of perillic acid in engineered microorganisms. The results also showed that other chemicals may be efficiently produced in C. tropicalis.Key points• Key genes cytoplasmic expression was conducive to L-limonene production in C. tropicalis.• Perillic acid was first synthesized de novo in engineered microorganisms.• The titer of perillic acid reached 106.69 mg/L in a 5-L fermenter.

RNAi targeting Nav and CPR via leaf delivery reduces adult emergence and increases the susceptibility to λ-cyholthin in Tuta absoluta (Meyrick)

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 (Nav) 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 TaNav 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 TaNav 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 LC30 doses of λ-cyholthin showed that silencing TaNav and TaCPR increased T. absoluta mortality about 32.2 and 17.4%, respectively, thus indicating that RNAi targeting TaNav and TaCPR could increase the susceptibility to λ-cyholthin in T. absoluta. This study demonstrates the potential of using RNAi targeting key genes, like TaNav and TaCPR, as an alternative technology for the control of this most destructive tomato pests in the future.

The Dissociation Process of NADP+ from NADPH-Cytochrome P450 Reductase Studied by Molecular Dynamics Simulation.

NADPH-cytochrome P450 reductase (CPR) plays a vital role as a redox partner for mammalian cytochrome P450 enzymes (P450s), facilitating the transfer of two electrons from NADPH to the P450 heme center in a sequential manner. Previous experimental studies revealed substantial domain movements of CPR, transitioning between closed and open states during the electron transfer (ET) cycle. These transitions are essential and are influenced by the binding of NADPH or the release of NADP+. However, the intricate molecular mechanisms governing the CPR-mediated ET cycle have largely remained elusive. This study employed molecular dynamics (MD) simulation techniques to explore the dissociation of NADP+ from CPR, a crucial step preceding the initial ET from CPR to a P450. Alongside the binding structure of NADP+ observed in a crystal structure (pose I), our MD simulations identified an alternative binding structure (pose II). Although pose II exhibits slightly lower stability than pose I, it can be formed through an approximate 210° counterclockwise rotation of the adenine group, with a free energy barrier of only 2.76 kcal/mol. The simulation results further suggest that NADP+ dissociation involves a tentative formation of pose II from pose I before complete dissociation, and that the binding of NADP+ to CPR is primarily governed by nonbonded interactions within the adenosine binding pocket.

A flexible linker of 8-amino acids between the membrane binding segment and the FMN domain of cytochrome P450 reductase is necessary for optimal activity

The diflavin NADPH-cytochrome P450 reductase (CYPOR) plays a critical role in human cytochrome P450 (CYP) activity by sequentially delivering two electrons from NADPH to CYP enzymes during catalysis. Although electron transfer to forty-eight human CYP enzymes by the FMN hydroquinone of CYPOR is well-known, the role of the linker between the NH2-terminus membrane-binding domain (MBD) and FMN domain in supporting the activity of P450 enzymes remains poorly understood. Here we demonstrate that a linker with at least eight residues is required to form a functional CYPOR-CYP2B4 complex. The linker has been shortened in two amino-acid increments from Phe44 to Ile57 using site directed mutagenesis. The ability of the deletion mutants to support cytochrome P450 2B4 (CYP2B4) catalysis and reduce ferric CYP2B4 was determined using an in vitro assay and stopped-flow spectrophotometry. Steady-state enzyme kinetics showed that shortening the linker by 8–14 amino acids inhibited (63–99%) the ability of CYPOR to support CYP2B4 activity and significantly increased the Km of CYPOR for CYP2B4. In addition, the reductase mutants decreased the rate of reduction of ferric CYP2B4 (46–95%) compared to wildtype when the linker was shortened by 8–14 residues. These results indicate that a linker with a minimum length of eight residues is necessary to enable the FMN domain of reductase to interact with CYP2B4 to form a catalytically competent complex. Our study provides evidence that the length of the MBD-FMN domain linker is a major determinant of the ability of CYPOR to support CYP catalysis and drug metabolism by P450 enzymes. PreambleThis manuscript is dedicated in memory of Dr. James R. Kincaid who was the doctoral advisor to Dr. Freeborn Rwere and a longtime collaborator and friend of Dr. Lucy Waskell. Dr. James R. Kincaid was a distinguished professor of chemistry specializing in resonance Raman (rR) studies of heme proteins. He inspired Dr. Rwere (a Zimbabwean native) and three other Zimbabweans (Dr. Remigio Usai, Dr. Daniel Kaluka and Ms. Munyaradzi E. Manyumwa) to use lasers to document subtle changes occurring at heme active site of globin proteins (myoglobin and hemoglobin) and cytochrome P450 enzymes. Dr. Rwere appreciate his contributions to the development of talented Black scientists from Africa.

Evaluation of lipopeptides biosurfactant from Raoultella planticola for bioremediation in n-hexadecane-contaminated soil

Biosurfactants combined with bioaugmentation for the remediation of petroleum hydrocarbon-contaminated soils proved to be one of the most effective ways. However, research on lipopeptides combined with bioaugmentation is rare, and the biological mechanisms are undefined. Therefore, the effect of Raoultella planticola and the lipopeptides it produces to restore contaminated soils was investigated using n-hexadecane as a model of petroleum hydrocarbons. After 60 d, n-hexadecane removal rate increased from 31.3% in contaminated soil to 39.9% with R. planticola alone and 59.0% with R. planticola together with 1.0 g intracellular lipopeptide (INLS) and 7.5 g sodium gluconate (SG)/kg dry soil. Meanwhile, the nitrogen and phosphorus components of the soil increased by 13.9% and 8% in the experimental group, respectively. Besides, DHA activity was 6.7 times that of the control group at 28 d, indicating a considerable increase in DHA activity. Microbial community analysis revealed a considerable increase in functional genera of Sphingomonas, Nocardia, Pseudoalteromonas, Povalibacter, Apiotrichum, Pseudallecheria, and Ascodesmis. The amount of fungus in the experimental group was more than 30 times that of the control group after 60 d. Microbial co-occurrence networks showed improved interactions of Firmicutes and Ascomycota, encouraging synergistic cooperation among microbes and thereby accelerating n-hexadecane breakdown. At once, the metabolic pathways ko00071 and ko00980 for n-hexadecane degradation were greatly elevated, indicating that functional genes such as alkane 1-monooxygenase and NADPH-cytochrome P450 reductase were activated. These results implied that combining hydrocarbon-degrading microbial with INLS was a promising bioremediation technique to remove soil petroleum hydrocarbons.

Mining and functional characterization of NADPH-cytochrome P450 reductases of the DNJ biosynthetic pathway in mulberry leaves

Background1-Deoxynojirimycin (DNJ), the main active ingredient in mulberry leaves, with wide applications in the medicine and food industries due to its significant functions in lowering blood sugar, and lipids, and combating viral infections. Cytochrome P450 is a key enzyme for DNJ biosynthesis, its activity depends on the electron supply of NADPH-cytochrome P450 reductases (CPRs). However, the gene for MaCPRs in mulberry leaves remains unknown.ResultsIn this study, we successfully cloned and functionally characterized two key genes, MaCPR1 and MaCPR2, based on the transcriptional profile of mulberry leaves. The MaCPR1 gene comprised 2064 bp, with its open reading frame (ORF) encoding 687 amino acids. The MaCPR2 gene comprised 2148 bp, and its ORF encoding 715 amino acids. The phylogenetic tree indicates that MaCPR1 and MaCPR2 belong to Class I and Class II, respectively. In vitro, we found that the recombinant enzymes MaCPR2 protein could reduce cytochrome c and ferricyanide using NADPH as an electron donor, while MaCPR1 did not. In yeast, heterologous co-expression indicates that MaCPR2 delivers electrons to MaC3'H hydroxylase, a key enzyme catalyzing the production of chlorogenic acid from 3-O-p-coumaroylquinic acid.ConclusionsThese findings highlight the orchestration of hydroxylation process mediated by MaCPR2 during the biosynthesis of secondary metabolite biosynthesis in mulberry leaves. These results provided a foundational understanding for fully elucidating the DNJ biosynthetic pathway within mulberry leaves.

RNA interference of NADPH[sbnd]cytochrome P450 increased deltamethrin susceptibility in a resistant strain of the Chagas disease vector Triatoma infestans

The enzyme NADPH-cytochrome P450 reductase (CPR) plays a central role in cytochromes P450 activity. Gene expression analysis of cytochromes P450 and CPR in deltamethrin-resistant and susceptible populations revealed that P450s genes are involved in the development of insecticide resistance in Triatoma infestans. To clarify the role of cytochromes P450 in insecticide resistance, it was proposed to investigate the effect of CPR gene silencing by RNA interference (RNAi) in a pyrethroid resistant population of T. infestans. Silencing of the CPR gene showed a significant increase in susceptibility to deltamethrin in the population analysed. This result support the hypothesis that the metabolic process of detoxification mediated by cytochromes P450 contributes to the decreased deltamethrin susceptibility observed in the resistant strain of T. infestans.

Heterologous expression in E. coli and functional characterization of the tomato CPR enzymes

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.

Identification of a Gene Encoding a New Fungal Steroid 7-Hydroxylase and Its Functional Characterization in Pichia pastoris Yeast.

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 (P450cur) 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 P450cur gene was increased more than 300-fold. A two-gene construct with a candidate P450cur 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 P450cur and CPR genes were obtained. The functional activity of the recombinant P450cur 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 P450cur 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.

Comparative gut proteomics study revealing adaptive physiology of Eurasian spruce bark beetle, Ips typographus (Coleoptera: Scolytinae)

The bark beetle, Ips typographus (L.), is a major pest of Norway spruce, Picea abies (L.), causing enormous economic losses globally. The adult stage of the I. typographus has a complex life cycle (callow and sclerotized); the callow beetles feed ferociously, whereas sclerotized male beetles are more aggressive and pioneers in establishing new colonies. We conducted a comparative proteomics study to understand male and female digestion and detoxification processes in callow and sclerotized beetles. Proteome profiling was performed using high-throughput liquid chromatography-mass spectrometry. A total of >3000 proteins were identified from the bark beetle gut, and among them, 539 were differentially abundant (fold change ±2, FDR <0.05) between callow and sclerotized beetles. The differentially abundant proteins (DAPs) mainly engage with binding, catalytic activity, anatomical activity, hydrolase activity, metabolic process, and carbohydrate metabolism, and hence may be crucial for growth, digestion, detoxification, and signalling. We validated selected DAPs with RT-qPCR. Gut enzymes such as NADPH-cytochrome P450 reductase (CYC), glutathione S-transferase (GST), and esterase (EST) play a crucial role in the I. typographus for detoxification and digesting of host allelochemicals. We conducted enzyme activity assays with them and observed a positive correlation of CYC and GST activities with the proteomic results, whereas EST activity was not fully correlated. Furthermore, our investigation revealed that callow beetles had an upregulation of proteins associated with juvenile hormone (JH) biosynthesis and chitin metabolism, whereas sclerotized beetles exhibited an upregulation of proteins linked to fatty acid metabolism and the TCA cycle. These distinctive patterns of protein regulation in metabolic and functional processes are specific to each developmental stage, underscoring the adaptive responses of I. typographicus in overcoming conifer defences and facilitating their survival. Taken together, it is the first gut proteomic study comparing males and females of callow and sclerotized I. typographus, shedding light on the adaptive ecology at the molecular level. Furthermore, the information about bark beetle handling of nutritionally limiting and defence-rich spruce phloem diet can be utilized to formulate RNAi-mediated beetle management.

Cloning and Functional Characterization of NADPH-Cytochrome P450 Reductases in Aconitum vilmorinianum

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.

Metabolism of testosterone and progesterone by cytochrome P450 2C19 allelic variants.

CYP2C19 is a member of the human microsomal cytochrome P450 (CYP). Significant variation in CYP2C19 levels and activity can be attributed to polymorphisms in this gene. Wildtype CYP2C19 and 13 mutants (CYP2C19.1B, CYP2C19.5A, CYP2C19.5B, CYP2C19.6, CYP2C19.8, CYP2C19.9, CYP2C19.10, CYP2C19.11, CYP2C19.13, CYP2C19.16, CYP2C19.19, CYP2C19.23, CYP2C19.30, and CYP2C19.33) were coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. Hydroxylase activity toward testosterone and progesterone was also examined. Ten CYP2C19 variants showed Soret peaks (450nm) typical of P450 in the reduced CO-difference spectra. CYP2C19.11 and CYP2C19.23 showed higher testosterone 11α, 16α-/17- and progesterone 6β-,21-,16α-/17α-hydroxylase activities than CYP2C19.1B. CYP2C19.6, CYP2C19.16, CYP2C19.19, and CYP2C19.30 showed lower activity than CYP2C19.1B. CYP2C19.9, CYP2C19.10. CYP2C19.13, and CYP2C19.33 showed different hydroxylation activities than CYP2C19.1B. These results indicated that CYP2C19 variants have very different substrate specificities for testosterone and progesterone.

RNA interference of NADPH-cytochrome P450 reductase increases the susceptibility of Aphis gossypii Glover to sulfoxaflor

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.

Class I and II NADPH-cytochrome P450 reductases exhibit different roles in triterpenoid biosynthesis in Lotus japonicus.

Cytochrome P450 monooxygenases (CYPs) are enzymes that play critical roles in the structural diversification of triterpenoids. To perform site-specific oxidations of the triterpene scaffold, CYPs require electrons transferred by NADPH-cytochrome P450 reductase (CPR), which is classified into two main classes, class I and class II, based on their structural difference. Lotus japonicus is a triterpenoids-producing model legume with one CPR class I gene (LjCPR1) and a minimum of two CPR class II genes (LjCPR2-1 and LjCPR2-2). CPR classes I and II from different plants have been reported to be involved in different metabolic pathways. By performing gene expression analyses of L. japonicus hairy root culture treated with methyl jasmonate (MeJA), this study revealed that LjCPR1, CYP716A51, and LUS were down-regulated which resulted in no change in betulinic acid and lupeol content. In contrast, LjCPR2s, bAS, CYP93E1, and CYP72A61 were significantly upregulated by MeJA treatment, followed by a significant increase of the precursors for soyasaponins, i.e. β-amyrin, 24-OH β-amyrin, and sophoradiol content. Triterpenoids profile analysis of LORE1 insertion and hairy root mutants showed that the loss of the Ljcpr2-1 gene significantly reduced soyasaponins precursors but not in Ljcpr1 mutants. However, Ljcpr1 and Ljcpr2-1 mutants showed a significant reduction in lupeol and oleanolic, ursolic, and betulinic acid contents. Furthermore, LjCPR1, but not LjCPR2, was crucial for seed development, supporting the previous notion that CPR class I might support plant basal metabolism. This study suggests that CPR classes I and II play different roles in L. japonicus triterpenoid biosynthesis.

Allosteric modulation of cytochrome P450 enzymes by the NADPH cytochrome P450 reductase FMN-containing domain

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.

Functional characterization of CYP1 enzymes: Complex formation, membrane localization and function

CYP1A1, CYP1A2, and CYP1B1 have a high degree of sequence similarity, similar substrate selectivities and induction characteristics. However, experiments suggest that there are significant differences in their quaternary structures and function. The goal of this study was to characterize the CYP1 proteins regarding their ability to form protein-protein complexes, lipid microdomain localization, and ultimately function. This was accomplished by examining (1) substrate metabolism of the CYP1s as a function of NADPH-cytochrome P450 reductase (POR) concentration, and (2) quaternary structure, using bioluminescence resonance energy transfer (BRET). Both CYP1As were able to form BRET-detectable homomeric complexes, which was not observed with CYP1B1. When activities were measured as a function of [POR], CYP1A1 and CYP1B1 showed a hyperbolic response, consistent with mass-action binding; however, CYP1A2 produced a sigmoidal response, suggesting that the homomeric complex affected its function. Differences were observed in their ability to form heteromeric complexes. Whereas CYP1B1 and CYP1A1 formed a complex, neither the CYP1A1/CYP1A2 nor the CYP1B1/CYP1A2 pair formed BRET-detectable complexes. These proteins also differed in their lipid microdomain localization, with CYP1A2 and CYP1B1 residing in ordered membranes, and CYP1A1 in the disordered lipid regions. Taken together, despite their sequence similarities, there are substantial differences in quaternary structures and microdomain localization that can influence enzymatic activities. As these proteins exist in the endoplasmic reticulum with other ER-resident proteins, the P450s need to be considered as part of multi-enzyme systems rather than simply monomeric proteins interacting with their redox partners.

Promoting Photosynthetic Production of Dammarenediol-II in Chlamydomonas reinhardtii via Gene Loading and Culture Optimization.

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.