Unraveling the role of cytochrome P450 enzymes in oleanane triterpenoid biosynthesis in arjuna tree.

Triterpenoids (C30-isoprenoids) represent a major group of natural products with various physiological functions in plants. Triterpenoids and their derivatives have medicinal uses owing to diverse bioactivities. Arjuna (Terminalia arjuna) tree bark accumulates highly oxygenated ß-amyrin-derived oleanane triterpenoids (e.g., arjunic acid, arjungenin, and arjunolic acid) with cardioprotective roles. However, biosynthetic routes and enzymes remain poorly understood. We mined the arjuna transcriptome and conducted cytochrome P450 monooxygenase (P450) assays using Saccharomyces cerevisiae and Nicotiana benthamiana to identify six P450s and two P450 reductases for oxidative modifications of oleanane triterpenoids. P450 assays using oleananes revealed a greater substrate promiscuity of C-2α and C-23 hydroxylases/oxidases than C-28 oxidases. CYP716A233 and CYP716A432 catalyzed ß-amyrin/erythrodiol C-28 oxidation to produce oleanolic acid. C-2α hydroxylases (CYP716C88 and CYP716C89) converted oleanolic acid and hederagenin to maslinic acid and arjunolic acid. CYP716C89 also hydroxylated erythrodiol and oleanolic aldehyde. However, CYP714E107a and CYP714E107b catalyzed oleanolic acid/maslinic acid/arjunic acid, C-23 hydroxylation to form hederagenin, arjunolic acid and arjungenin, and hederagenin C-23 oxidation to produce gypsogenic acid, but at a lower rate than oleanolic acid C-23 hydroxylation. Overall, P450 substrate selectivity suggested that C-28 oxidation is the first P450-catalyzed oxidative modification in the arjuna triterpenoid pathway. However, the pathway might branch thereafter through C-2α/C-23 hydroxylation of oleanolic acid. Taken together, these results provided new insights into substrate range of P450s and unraveled biosynthetic routes of triterpenoids in arjuna. Moreover, complete elucidation and reconstruction of arjunolic acid pathway in S. cerevisiae and N. benthamiana suggested the utility of arjuna P450s in heterologous production of cardioprotective compounds.

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.

Delayed gametocyte clearance in Plasmodium vivax malaria is associated with polymorphisms in the cytochrome P450 reductase (CPR).

Primaquine (PQ) is the main drug used to eliminate dormant liver stages and prevent relapses in Plasmodium vivax malaria. It also has an effect on the gametocytes of Plasmodium falciparum; however, it is unclear to what extent PQ affects P. vivax gametocytes. PQ metabolism involves multiple enzymes, including the highly polymorphic CYP2D6 and the cytochrome P450 reductase (CPR). Since genetic variability can impact drug metabolism, we conducted an evaluation of the effect of CYP2D6 and CPR variants on PQ gametocytocidal activity in 100 subjects with P. vivax malaria. To determine gametocyte density, we measured the levels of pvs25 transcripts in samples taken before treatment (D0) and 72 hours after treatment (D3). Generalized estimating equations (GEEs) were used to examine the effects of enzyme variants on gametocyte densities, adjusting for potential confounding factors. Linear regression models were adjusted to explore the predictors of PQ blood levels measured on D3. Individuals with the CPR mutation showed a smaller decrease in gametocyte transcript levels on D3 compared to those without the mutation (P = 0.02, by GEE). Consistent with this, higher PQ blood levels on D3 were associated with a lower reduction in pvs25 transcripts. Based on our findings, the CPR variant plays a role in the persistence of gametocyte density in P. vivax malaria. Conceptually, our work points to pharmacogenetics as a non-negligible factor to define potential host reservoirs with the propensity to contribute to transmission in the first days of CQ-PQ treatment, particularly in settings and seasons of high Anopheles human-biting rates.

Functional and structural insight into the flexibility of cytochrome P450 reductases from Sorghum bicolor and its implications for lignin composition.

Plant NADPH-dependent cytochrome P450 reductase (CPR) is a multidomain enzyme that donates electrons for hydroxylation reactions catalyzed by class II cytochrome P450 monooxygenases involved in the synthesis of many primary and secondary metabolites. These P450 enzymes include trans-cinnamate-4-hydroxylase, p-coumarate-3'-hydroxylase, and ferulate-5-hydroxylase involved in monolignol biosynthesis. Because of its role in monolignol biosynthesis, alterations in CPR activity could change the composition and overall output of lignin. Therefore, to understand the structure and function of three CPR subunits from sorghum, recombinant subunits SbCPR2a, SbCPR2b, and SbCPR2c were subjected to X-ray crystallography and kinetic assays. Steady-state kinetic analyses demonstrated that all three CPR subunits supported the oxidation reactions catalyzed by SbC4H1 (CYP73A33) and SbC3'H (CYP98A1). Furthermore, comparing the SbCPR2b structure with the well-investigated CPRs from mammals enabled us to identify critical residues of functional importance and suggested that the plant flavin mononucleotide-binding domain might be more flexible than mammalian homologs. In addition, the elucidated structure of SbCPR2b included the first observation of NADP+ in a native CPR. Overall, we conclude that the connecting domain of SbCPR2, especially its hinge region, could serve as a target to alter biomass composition in bioenergy and forage sorghums through protein engineering.

The cytochrome P450 reductase CprA is a rate-limiting factor for Cyp51A-mediated azole resistance in Aspergillus fumigatus.

Azole antifungals remain the "gold standard" therapy for invasive aspergillosis. The world-wide emergence of isolates resistant to this drug class, however, developed into a steadily increasing threat to human health over the past years. In Aspergillus fumigatus, major mechanisms of resistance involve increased expression of cyp51A encoding one of two isoenzymes targeted by azoles. Yet, the level of resistance caused by cyp51A upregulation, driven by either clinically relevant tandem repeat mutations within its promoter or the use of high expressing heterologous promoters, is limited. Cytochrome P450 enzymes such as Cyp51A rely on redox partners that provide electrons for their activity. A. fumigatus harbors several genes encoding putative candidate proteins including two paralogous cytochrome P450 reductases, CprA and CprB, and the cytochrome b 5 CybE. In this work, we investigated the contribution of each cprA, cprB, and cybE overexpression to cyp51A-mediated resistance to different medical and agricultural azoles. Using the bidirectional promoter PxylP, we conditionally expressed these genes in combination with cyp51A, revealing cprA as the main limiting factor. Similar to this approach, we overexpressed cprA in an azole-resistant background strain carrying a cyp51A allele with TR34 in its promoter, which led to a further increase in its resistance. Employing sterol measurements, we demonstrate an enhanced eburicol turnover during upregulation of either cprA or cyp51A, which was even more pronounced during their simultaneous overexpression. In summary, our work suggests that mutations leading to increased Cyp51A activity through increased electron supply could be key factors that elevate azole resistance.

POR overexpression induces tamoxifen-resistance in breast cancer through the STAT1/c-Myc pathway.

Breast cancer is the most common cancer in women worldwide. Although tamoxifen (TAM), a selective estrogen receptor (ER) modulator, is widely used to treat ER-positive breast cancers, resistance to TAM remains a major clinical problem. NADPH-dependent cytochrome P450 reductase (POR) is known to participate in drug metabolism and steroid metabolism. Recent studies showed that high POR expression was correlated with poor outcomes in triple-negative breast cancer (TNBC), and POR might be a prognostic biomarker in TNBC. However, the role of POR in TAM resistance is still elusive. In this study, we found that high POR expression was associated with poor prognosis of ER-positive and TAM-treated breast cancer patients. In addition, COX analysis showed that POR expression was an independent prognostic biomarker for ER-positive as well as TAM-treated breast cancer patients. Furthermore, our results suggested that POR overexpression promoted TAM resistance by activating the STAT1/c-Myc pathway in ER-positive breast cancer cells. Immunohistochemical analysis showed that high POR/STAT1 expression was correlated with poor prognosis in TAM-treated breast cancer patients. Notably, combined treatment with TAM and a specific STAT1 inhibitor Fludarabine was more effective for inhibiting TAM-resistant breast cancer cells. Altogether, our findings suggested that POR overexpression induced TAM resistance through STAT1/c-Myc pathway and might serve as an independent prognostic biomarker in TAM-treated breast cancer patients. Combining TAM and STAT1 inhibitors might be an effective strategy for treating POR-induced TAM-resistant breast cancer.

Prokaryotic expression and characterization of artificial self-sufficient CYP120A monooxygenases.

Cytochrome P450 monooxygenases CYP120As are the unique non-membrane P450s, which are extensively involved in retinoid biodegradation. As the O-functionalized 1,3,3-trimethylcyclohex-1-ene moiety exists in many bioactive compounds which could only be catalyzed by Class II P450s, exploration of the catalytic repertoire of CYP120As is therefore highly attractive. However, up to date, only one bacteriogenic candidate (CYP120A1) was demonstrated for the hydroxylation of C16 and C17 of retinoic acid, by utilizing the integral membrane protein cytochrome P450 reductase redox partner for the electron transfer. Herein, we provided an efficient prokaryotic functional expression system of CYP120As in E. coli by expression of the CYP120A1 coupled with several reductase partners. Fusion redox partners to the C-terminal of the heme-domain are also working on other CYP120A members. Among them, the fusion protein of CYP120A29 and FAD/FMN reductase from Bacillus megaterium P450BM3 (CYP101A2) showed the highest expression level. Based on the available translational fusion systems, the regioselectivity and the substrate scope of the CYP120As have also been explored. This work represents a good starting point for further expanding the catalytic potential of CYP120 family. KEY POINTS: • Characterization of CYP120As in E. coli is firstly achieved by constructing fusion proteins. • The feasibility of three P450 reductase domains to CYP120As was evaluated. • Hydroxylated products of retinoic acid by six CYP120As were sorted and analyzed.

A Novel, Highly Potent NADPH-Dependent Cytochrome P450 Reductase from Waste Liza klunzingeri Liver.

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

NADPH-cytochrome P450 reductase knockdown decreases the response to precocene I in the migratory locust Locusta migratoria.

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.

Overexpression of NADPH-cytochrome P450 reductase is associated with sulfoxaflor resistance and neonicotinoid cross-resistance in Nilaparvata lugens (Stål).

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.

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.

Heme oxygenase-1 affects cytochrome P450 function through the formation of heteromeric complexes: Interactions between CYP1A2 and heme oxygenase-1.

Heme oxygenase 1 (HO-1) and the cytochromes P450 (P450s) are endoplasmic reticulum-bound enzymes that rely on the same protein, NADPH-cytochrome P450 reductase (POR), to provide the electrons necessary for substrate metabolism. Although the HO-1 and P450 systems are interconnected owing to their common electron donor, they generally have been studied separately. As the expressions of both HO-1 and P450s are affected by xenobiotic exposure, changes in HO-1 expression can potentially affect P450 function and, conversely, changes in P450 expression can influence HO-1. The goal of this study was to examine interactions between the P450 and HO-1 systems. Using bioluminescence resonance energy transfer (BRET), HO-1 formed HO-1•P450 complexes with CYP1A2, CYP1A1, and CYP2D6, but not all P450s. Studies then focused on the HO-1-CYP1A2 interaction. CYP1A2 formed a physical complex with HO-1 that was stable in the presence of POR. As expected, both HO-1 and CYP1A2 formed BRET-detectable complexes with POR. The POR•CYP1A2 complex was readily disrupted by the addition of HO-1, whereas the POR•HO-1 complex was not significantly affected by the addition of CYP1A2. Interestingly, enzyme activities did not follow this pattern. BRET data suggested substantial inhibition of CYP1A2-mediated 7-ethoxyresorufin de-ethylation in the presence of HO-1, whereas its activity was actually stimulated at subsaturating POR. In contrast, HO-1-mediated heme metabolism was inhibited at subsaturating POR. These results indicate that HO-1 and CYP1A2 form a stable complex and have mutual effects on the catalytic behavior of both proteins that cannot be explained by a simple competition for POR.

NADPH-Cytochrome P450 Reductase Mediates the Fatty Acid Desaturation of ω3 and ω6 Desaturases from Mortierella alpina.

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.

Stimulation of de novo glutathione synthesis by nitrofurantoin for enhanced resilience of hepatocytes.

Toxicity is not only a function of damage mechanisms, but is also determined by cellular resilience factors. Glutathione has been reported as essential element to counteract negative influences. The present work hence pursued the question how intracellular glutathione can be elevated transiently to render cells more resistant toward harmful conditions. The antibiotic nitrofurantoin (NFT) was identified to stimulate de novo synthesis of glutathione in the human hepatoma cell line, HepG2, and in primary human hepatocytes. In intact cells, activation of NFT yielded a radical anion, which subsequently initiated nuclear-factor-erythroid 2-related-factor-2 (Nrf2)-dependent induction of glutamate cysteine ligase (GCL). Application of siRNA-based intervention approaches confirmed the involvement of the Nrf2-GCL axis in the observed elevation of intracellular glutathione levels. Quantitative activation of Nrf2 by NFT, and the subsequent rise in glutathione, were similar as observed with the potent experimental Nrf2 activator diethyl maleate. The elevation of glutathione levels, observed even 48 h after withdrawal of NFT, rendered cells resistant to different stressors such as the mitochondrial inhibitor rotenone, the redox cycler paraquat, the proteasome inhibitors MG-132 or bortezomib, or high concentrations of NFT. Repurpose of the antibiotic NFT as activator of Nrf2 could thus be a promising strategy for a transient and targeted activation of the endogenous antioxidant machinery. Graphical abstract.

Identification of the contact region responsible for the formation of the homomeric CYP1A2•CYP1A2 complex.

Previous studies showed that cytochrome P450 1A2 (CYP1A2) forms a homomeric complex that influences its metabolic characteristics. Specifically, CYP1A2 activity exhibits a sigmoidal response as a function of NADPH-cytochrome P450 reductase (POR) concentration and is consistent with an inhibitory CYP1A2•CYP1A2 complex that is disrupted by increasing [POR] (Reed et al. (2012) Biochem. J. 446, 489-497). The goal of this study was to identify the CYP1A2 contact regions involved in homomeric complex formation. Examination of X-ray structure of CYP1A2 implicated the proximal face in homomeric complex formation. Consequently, the involvement of residues L91-K106 (P1 region) located on the proximal face of CYP1A2 was investigated. This region was replaced with the homologous region of CYP2B4 (T81-S96) and the protein was expressed in HEK293T/17 cells. Complex formation and its disruption was observed using bioluminescence resonance energy transfer (BRET). The P1-CYP1A2 (CYP1A2 with the modified P1 region) exhibited a decreased BRET signal as compared with wild-type CYP1A2 (WT-CYP1A2). On further examination, P1-CYP1A2 was much less effective at disrupting the CYP1A2•CYP1A2 homomeric complex, when compared with WT-CYP1A2, thereby demonstrating impaired binding of P1-CYP1A2 to WT-CYP1A2 protein. In contrast, the P1 substitution did not affect its ability to form a heteromeric complex with CYP2B4. P1-CYP1A2 also showed decreased activity as compared with WT-CYP1A2, which was consistent with a decrease in the ability of P1-CYP1A2 to associate with WT-POR, again implicating the P1 region in POR binding. These results indicate that the contact region responsible for the CYP1A2•CYP1A2 homomeric complex resides in the proximal region of the protein.

Heteromeric complex formation between human cytochrome P450 CYP1A1 and heme oxygenase-1.

P450 and heme oxygenase-1 (HO-1) receive their necessary electrons by interaction with the NADPH-cytochrome P450 reductase (POR). As the POR concentration is limiting when compared with P450 and HO-1, they must effectively compete for POR to function. In addition to these functionally required protein-protein interactions, HO-1 forms homomeric complexes, and several P450s have been shown to form complexes with themselves and with other P450s, raising the question, 'How are the HO-1 and P450 systems organized in the endoplasmic reticulum?' Recently, CYP1A2 was shown to associate with HO-1 affecting the function of both proteins. The goal of this study was to determine if CYP1A1 formed complexes with HO-1 in a similar manner. Complex formation among POR, HO-1, and CYP1A1 was measured using bioluminescence resonance energy transfer, with results showing HO-1 and CYP1A1 form a stable complex that was further stabilized in the presence of POR. The POR•CYP1A1 complex was readily disrupted by the addition of HO-1. CYP1A1 also was able to affect the POR•HO-1 complex, although the effect was smaller. This interaction between CYP1A1 and HO-1 also affected function, where the presence of CYP1A1 inhibited HO-1-mediated bilirubin formation by increasing the KmPOR•HO-1 without affecting the Vmaxapp. In like manner, HO-1 inhibited CYP1A1-mediated 7-ethoxyresorufin dealkylation by increasing the KmPOR•CYP1A1. Based on the mathematical simulation, the results could not be explained by a model where CYP1A1 and HO-1 simply compete for POR, and are consistent with the formation of a stable CYP1A1•HO-1 complex that affected the functional characteristics of both moieties.

Cryo-EM reveals the architecture of the dimeric cytochrome P450 CYP102A1 enzyme and conformational changes required for redox partner recognition.

Cytochrome P450 family 102 subfamily A member 1 (CYP102A1) is a self-sufficient flavohemeprotein and a highly active bacterial enzyme capable of fatty acid hydroxylation at a >3,000 min-1 turnover rate. The CYP102A1 architecture has been postulated to be responsible for its extraordinary catalytic prowess. However, the structure of a functional full-length CYP102A1 enzyme remains to be determined. Herein, we used a cryo-EM single-particle approach, revealing that full-length CYP102A1 forms a homodimer in which both the heme and FAD domains contact each other. The FMN domain of one monomer was located close to the heme domain of the other monomer, exhibiting a trans configuration. Moreover, full-length CYP102A1 is highly dynamic, existing in multiple conformational states, including open and closed states. In the closed state, the FMN domain closely contacts the FAD domain, whereas in the open state, one of the FMN domains rotates away from its FAD domain and traverses to the heme domain of the other monomer. This structural arrangement and conformational dynamics may facilitate rapid intraflavin and trans FMN-to-heme electron transfers (ETs). Results with a variant having a 12-amino-acid deletion in the CYP102A1 linker region, connecting the catalytic heme and the diflavin reductase domains, further highlighted the importance of conformational dynamics in the ET process. Cryo-EM revealed that the Δ12 variant homodimer is conformationally more stable and incapable of FMN-to-heme ET. We conclude that closed-to-open alternation is crucial for redox partner recognition and formation of an active ET complex for CYP102A1 catalysis.

Functional expression of eukaryotic cytochrome P450s in yeast.

Cytochrome P450 enzymes (P450s) are a superfamily of heme-thiolate proteins widely existing in various organisms. Due to their key roles in secondary metabolism, degradation of xenobiotics, and carcinogenesis, there is a great demand to heterologously express and obtain a sufficient amount of active eukaryotic P450s. However, most eukaryotic P450s are endoplasmic reticulum-localized membrane proteins, which is the biggest challenge for functional expression to high levels. Furthermore, the functions of P450s require the cooperation of cytochrome P450 reductases for electron transfer. Great efforts have been devoted to the heterologous expression of eukaryotic P450s, and yeasts, particularly Saccharomyces cerevisiae are frequently considered as the first expression systems to be tested for this challenging purpose. This review discusses the strategies for improving the expression and activity of eukaryotic P450s in yeasts, followed by examples of P450s involved in biosynthetic pathway engineering.

NADPH-cytochrome P450 reductase potentially involved in indoxacarb resistance in Spodoptera litura.

NADPH-cytochrome P450 reductase (CPR) plays a central role in the metabolism of insecticides. Numerous studies have shown that CPR is associated with insecticide resistance in insect. In this study, two transcripts of Spodoptera litura CPR (SlCPR-X1 and SlCPR-X2) were identified and cloned, and the deduced protein of SlCPR-X1 contains all the conserved CPR structural features (N-terminal membrane anchor, FMN, FAD and NADP binding domains, FAD binding motif, and catalytic residues). However, no N-terminal member anchor and a shorter FMN binding region have been identified in the deduced protein of SlCPR-X2. The specific expression patterns showed that SlCPR-X1 and SlCPR-X2 were detected in all tested developmental stages and tissues, but highly expressed in third-, fourth-, and fifth-instar larvae, and in midgut and fat body. In addition, compared with the susceptible strain, SlCPR-X1 and SlCPR-X2 were up-regulated and more inducible when treated with indoxacarb in the indoxacarb-resistant strain. However, the relative expression, up-regulation and induction of SlCPR-X1 were all higher than those of SlCPR-X2 in the indoxacarb-resistant strain. Furthermore, RNA interference and baculovirus expression system combined with MTT cytotoxicity assay demonstrated that only SlCPR-X1 with the N-terminal membrane anchor as the major CPR potentially involved in S. litura indoxacarb resistance. The outcome of this study further expands our understanding of the important role of insect CPR in xenobiotics detoxification and resistance development, and CPR could be a potential target for insecticide resistance management mediated by RNAi or CRISPR/Cas.

Conformational Landscape of Cytochrome P450 Reductase Interactions.

Oxidative reactions catalyzed by Cytochrome P450 enzymes (CYPs), which constitute the most relevant group of drug-metabolizing enzymes, are enabled by their redox partner Cytochrome P450 reductase (CPR). Both proteins are anchored to the membrane of the endoplasmic reticulum and the CPR undergoes a conformational change in order to interact with the respective CYP and transfer electrons. Here, we conducted over 22 microseconds of molecular dynamics (MD) simulations in combination with protein-protein docking to investigate the conformational changes necessary for the formation of the CPR-CYP complex. While some structural features of the CPR and the CPR-CYP2D6 complex that we highlighted confirmed previous observations, our simulations revealed additional mechanisms for the conformational transition of the CPR. Unbiased simulations exposed a movement of the whole protein relative to the  membrane, potentially to facilitate interactions with its diverse set of redox partners. Further, we present a structural mechanism for the susceptibility of the CPR to different redox states based on the flip of a glycine residue disrupting the local interaction network that maintains inter-domain proximity. Simulations of the CPR-CYP2D6 complex pointed toward an additional interaction surface of the FAD domain and the proximal side of CYP2D6. Altogether, this study provides novel structural insight into the mechanism of CPR-CYP interactions and underlying conformational changes, improving our understanding of this complex machinery Cytochrome P450 reductase; CPR; conformational; dynamicsrelevant for drug metabolism.