NADPH-dependent Cytochrome P450 Reductase Research Articles

Cytochrome b5 diversity in green lineages preceded the evolution of syringyl lignin biosynthesis.

Lignin production marked a milestone in vascular plant evolution, and the emergence of syringyl (S)-lignin is lineage-specific. S-lignin biosynthesis in angiosperms, mediated by ferulate 5-hydroxylase (F5H, CYP84A1), has been considered a recent evolutionary event. F5H uniquely requires the cytochrome b5 protein CB5D as an obligatory redox partner for catalysis. However, it remains unclear how CB5D functionality originated and whether it co-evolved with F5H. We reveal here the ancient evolution of CB5D-type function supporting F5H-catalyzed S-lignin biosynthesis. CB5D emerged in charophyte algae, the closest relatives of land plants, and is conserved and proliferated in embryophytes, especially in angiosperms, suggesting functional diversification of the CB5 family before terrestrialization. A sequence motif containing acidic amino residues in helix 5 of the CB5 heme-binding domain contributes to the retention of CB5D function in land plants but not in algae. Notably, CB5s in the S-lignin-producing lycophyte Selaginella lack these residues, resulting in no CB5D-type function. An independently evolved S-lignin biosynthetic F5H (CYP788A1) in Selaginella relies on NADPH-dependent cytochrome P450 reductase as sole redox partner, distinct from angiosperms. These results suggest that angiosperm F5Hs co-opted the ancient CB5D, forming a modern cytochrome P450 monooxygenase system for aromatic ring meta-hydroxylation, enabling the re-emergence of S-lignin biosynthesis in angiosperms.

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.

Target-site and metabolic mechanisms of tolerance to penoxsulam in pond lovegrass (Eragrostis japonica)

AbstractThe identification of herbicide tolerance is essential for effective chemical weed control. According to whole-plant dose–response assays, none of 29 pond lovegrass [Eragrostis japonica (Thunb.) Trin.] populations were sensitive to penoxsulam. The effective dose values of penoxsulam causing 50% inhibition of fresh weight (GR50: 105.14 to 148.78 g ai ha−1) in E. japonica populations were much higher than the label rate of penoxsulam (15 to 30 g ai ha−1) in the field. This confirmed that E. japonica was tolerant to penoxsulam. Eragrostis japonica populations showed 52.83- to 74.76-fold higher tolerance to penoxsulam than susceptible barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.]. The mechanisms of tolerance to penoxsulam in E. japonica were also identified. In vitro activity assays revealed that the penoxsulam concentration required to inhibit 50% of the acetolactate synthase (ALS) activity (IC50) was 12.27-fold higher in E. japonica than in E. crus-galli. However, differences in the ALS gene, previously found to endow target-site resistance in weeds, were not detected in the sequences obtained. Additionally, the expression level of genes encoding ALS in E. japonica was approximately 2-fold higher than in E. crus-galli after penoxsulam treatment. Furthermore, penoxsulam tolerance can be significantly reversed by three cytochrome P450 monooxygenase (CytP450) inhibitors (1-aminobenzotriazole, piperonyl butoxide, and malathion), and the activity of NADPH-dependent cytochrome P450 reductase toward penoxsulam in E. japonica increased significantly (approximately 7-fold higher) compared with that of treated E. crus-galli. Taken together, these results indicate that lower ALS sensitivity, relatively higher ALS expression levels, and stronger metabolism of CytP450s combined to bring about penoxsulam tolerance in E. japonica.

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

Breast cancer is the most common cancer in women worldwide. Althoughtamoxifen (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.

АНАЛИЗ АКТИВНОСТИ МИКРОСОМАЛЬНЫХ РЕДУКТАЗ ТКАНИ ЯИЧНИКОВ ПОСЛЕ КРИОКОНСЕРВАЦИИ МЕТОДОМ АКТИВИРОВАННОЙ ХЕМИЛЮМИНЕСЦЕНЦИИ

The aim of the study was to investigate the activity of NADH-dependent cytochrome b5 reductase (CYB5R) and NADPH-dependent cytochrome P450 reductase (CYPOR) in ovarian tissues after cryopreservation by lucigenin-enhanced chemiluminescence with NADH and NADPH stimulation, respectively. The results indicate that both mitochondrial and microsomal reductase activities are preserved in cryopreserved ovarian tissues. After cryopreservation, the level of production of superoxide anion radical by mitochondria drops by 3–10 times, while the presence or absence of chemotherapy has no effect, and this parameter is also not affected by the severity of the disease. Compared to the control group (patients with benign tumors), the activity of CYB5R and CYPOR of ovarian tissue in a malignant cancer process decreases 1.5–10 times depending on the disease severity, and the presence of chemotherapy dramatically affects these parameters — the activity of microsomal reductases decreases by 50–100 times in chemotherapy compared to the control group. Thus, microsomal reductase activity is a more informative parameter for assessing the functionality of cryopreserved ovarian tissue than superoxide-producing capacity of mitochondria, because, firstly, it depends on the stage of disease and prior chemotherapy, and secondly, the analytical signal of NADH/NADPH stimulated chemiluminescence is characterized by approximately 30 times higher intensity than mitochondrial chemiluminescence, which leads to higher analytical sensitivity of the technique.

Approaches for increasing the electrocatalitic efficiency of cytochrome P450 3A4

The electrochemically driven cytochrome P450 reactions have great promise as drug sensing device, new drug searching tool and bioreactor with broad synthetic application. In the present work, we proposed approaches for the increasing the efficiency of cytochrome P450 3A4 electrocatalysis, based on fine regulation and reproduction of nature hemeprotein catalytic cycle and electron transfer pathways on electrode. To analyze the comparative electrochemical and electrocatalytic activity, cytochrome P450 3A4 was immobilized on electrodes modified with a membrane-like synthetic surfactant, didodecyldimethylammonium bromide (DDAB). We used riboflavin, FMN and FAD as low molecular models of NADPH-dependent cytochrome P450 reductase for the improving and enhancement properties of catalytically responsible cytochrome P450 3A4-electrode. The efficiencies of electrocatalysis of erythromycin N-demethylation as well-known cytochrome P450 3A4 substrate in the case of riboflavin, FAD and FMN as electron transfer mediators were 135 ± 6, 171 ± 15 and 203 ± 10 %, respectively (in comparison with 100 ± 18 % erythromycin N-demethylation in the case of cytochrome P450 3A4-electrode as catalyst). Molecular modeling of cytochrome P450 3A4 complexes with riboflavin, FMN and FAD confirms possibility of binding isoalloxazine ring of riboflavin to the protein on the proximal side of hemeprotein, which is the place for binding of redox partners of the cytochrome P450.

Multivariate modular metabolic engineering for enhanced gibberellic acid biosynthesis in Fusarium fujikuroi

Gibberellic acid (GA3) is one of natural phytohormones, widely used in agriculture and downstream fields. Qualified for the nature productivity, Fusarium fujikuroi was currently employed for the industrial biotransformation from agriculture residues into GA3. Herein, Multivariate modular metabolic engineering (MMME) was assigned to reconstitute the metabolic balance in F. fujikuroi for enhancing GA3 production. Three modules including precursor pool, cluster-specific channel and P450-mediated oxidation in GA3 biosynthetic pathway were defined and optimized separately. The enhancement of both precursor pool and cluster-specific channel pushed metabolic flux transfer into the GA3-specific pathway. Moreover, both introduction of Vitreoscilla hemoglobin and reinforcement of NADPH-dependent cytochrome P450 reductase facilitated oxidation cofactor transfer and subsequently boosted mycelium growth and GA3 biosynthesis. Integration of three modules in the engineered strain accumulated 2.89 g/L GA3 in shake flask via submerged fermentation, presenting a promising modular metabolic engineering model for efficient microbial transformation in agro-industrial application.

Two mechanisms provide tolerance to cyhalofop-butyl in pond lovegrass [Eragrostis japonica (Thunb.) Trin.

Pond lovegrass [Eragrostis japonica (Thunb.) Trin.] is an annual grass weed of rice fields worldwide. Cyhalofop-butyl has been widely used for controlling annual grass weeds in rice fields. However, E. japonica is tolerant to cyhalofop-butyl. The effective dose values of cyhalofop-butyl for 29 E. japonica populations causing 50% inhibition of fresh weight (GR50: 130.15 to 187.61 g a.i. ha−1) were much higher than the recommended dose of cyhalofop-butyl (75 g a.i. ha−1) in the field. The mechanisms of tolerance to cyhalofop-butyl in E. japonica were identified. In vitro activity assays revealed that the cyhalofop-butyl concentration required to inhibit 50% of the acetyl-coenzyme A carboxylase (ACCase) activity (IC50) was 6.22-fold higher in E. japonica than that in the cyhalofop-butyl-susceptible Chinese sprangletop [Leptochloa chinensis (L.) Nees]. However, mutations in the ACCase gene, previously found to endow target-site resistance in weeds, were not detected in the sequences obtained. Additionally, the expression level of genes encoding ACCase in E. japonica was found to be as similar to L. chinensis. Tolerance was reduced by two cytochrome P450 monooxygenases (Cyt P450s) inhibitors (1-aminobenzotriazole and piperonyl butoxide) and the activity of NADPH-dependent cytochrome P450 reductase in E. japonica was approximately 4.46-fold higher than that of L. chinensis after cyhalofop-butyl treatment. Taken together, it is concluded that two co-existing mechanisms, an insensitive target ACCase and an enhanced metabolism mediated by Cyt P450s, endow tolerance to cyhalofop-butyl in E. japonica.

Functional characterization of CYP6G4 from the house fly in propoxur metabolism and resistance

The house fly (Musca domestica L.) (Diptera: Muscidae) is a global vector that can transmit >250 human and animal diseases. The control of house flies has heavily relied on the application of various chemical insecticides. The carbamate insecticide propoxur has been widely used for the control of house flies, and resistance to propoxur has been documented in many house fly populations worldwide. Previous studies have identified several propoxur resistance-conferring mutations in the target protein acetylcholinesterase; however, the molecular basis for metabolic resistance to propoxur remains unknown. In this study, we investigated the involvement of CYP6G4, a cytochrome P450 overexpressed in many insecticide resistant populations of Musca domestica, in propoxur metabolism and resistance by using combined approaches of recombinant protein-based insecticide metabolism and the Drosophila GAL4/UAS transgenic system. The recombinant CYP6G4 and its redox partners (NADPH-dependent cytochrome P450 reductase and cytochrome b5) were functionally expressed in Escherichia coli. Metabolism experiments showed that CYP6G4 was able to transform propoxur with a turnover rate of around 0.79 min−1. Six metabolites were putatively identified, suggesting that CYP6G4 could metabolize propoxur via hydroxylation, O-depropylation and N-demethylation. Moreover, bioassay results showed that ectopic overexpression of CYP6G4 in fruit flies significantly increased their tolerance to propoxur. Our in vivo and in vitro data convincingly demonstrate that CYP6G4 contributes to propoxur metabolism and resistance.

Biological and Pharmacological Properties of Carbon Monoxide: A General Overview

Carbon monoxide (CO) is one of the most common causes of inhalation poisoning worldwide. However, it is also well known that CO is produced endogenously in the heme degradation reaction catalyzed by heme oxygenase (HO) enzymes. HO catalyzes the degradation of heme to equimolar quantities of CO, iron ions (Fe2+), and biliverdin. Three oxygen molecules (O2) and the electrons provided by NADPH-dependent cytochrome P450 reductase are used in the reaction. HO enzymes comprise three distinct isozymes: the inducible form, heme oxygenase-1 (HO-1); the constitutively expressed isozyme, heme oxygenase-2 (HO-2); and heme oxygenase-3 (HO-3), which is ubiquitously expressed but possesses low catalytic activity. According to some authors, HO-3 is rather a pseudogene originating from the HO-2 transcript, and it has only been identified in rats. Therefore, cellular HO activity is provided by two major isoforms—the inducible HO-1 and the constitutively expressed HO-2. For many years, endogenously generated CO was treated as a by-product of metabolism without any serious physiological or biochemical significance, while exogenous CO was considered only as an extremely toxic gas with lethal effects. Research in recent years has proven that endogenous and exogenous CO (which may be surprising, given public perceptions) acts not only as an agent that affects many intracellular pathways, but also as a therapeutic molecule. Hence, the modulation of the HO/CO system may be one option for a potential therapeutic strategy. Another option is the administration of CO by exogenous inhalation. As alternatives to gas administration, compounds known as CO-releasing molecules (CORMs) can be administered, since they can safely release CO in the body. The aim of this article is to provide a brief overview of the physiological and biochemical properties of CO and its therapeutic potential.

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.

Pyrethrin-resembling pyrethroids are metabolized more readily than heavily modified ones by CYP9As from Helicoverpa armigera

The cotton bollworm, Helicoverpa armigera, is a polyphagous pest threatening many economically important crops worldwide. Until recently, synthetic pyrethroids remain in wide use for controlling pest insects including the cotton bollworm. Understanding the metabolic mechanism of pyrethroids in a given pest can provide significant implication for a smart choice of insecticides, and such information is useful for the development of novel selective and safe insecticides. In this study, we used complexes of recombinant H. armigera cytochrome P450 CYP9A and NADPH-dependent cytochrome P450 reductase to investigate the capacity of three CYP9A paralogs in the transformation of seven structurally different pyrethroids by metabolism assays. The results showed that the three paralogous CYP9As were able to metabolize multiple pyrethroids. Interestingly, all the three CYP9As transformed pyrethrin-resembling pyrethroids (e.g. bioallethrin) more efficiently than the heavily modified ones (e.g. bifenthrin). These findings suggest that herbivorous insects can cope with synthetic insecticides using their physiological systems that initially evolved to survive exposure to the defensive chemicals in their host plants, adding support to the pre-adaptation hypothesis.

Proteoliposomes as a Method of Membrane Protein Immobilization for SPR-analysis with the Human CYP3A4 and CYB5A Interaction as an Example

Microsomal systems of human cytochrome P450 consist of three components, which are membrane proteins: cytochrome P450 hemoprotein (CYP), NADPH-dependent cytochrome P450 reductase (CPR), and a small regulatory heme-containing protein cytochrome b5 (CYB5A). In the study of the cytochrome P450 system functioning the study of intermolecular interactions both with partner proteins and with possible drug prototypes is of great importance. Surface plasmon resonance (SPR) is a powerful and reliable tool for studying intermolecular interactions. However, there is a problem of immobilization of membrane proteins on the optical chip of the SPR biosensor. It is important to immobilize such proteins in native conditions with respect to the correct orientation of the protein globule to the surface of sensor. Previously, we have developed and described a method involving direct native immobilization of membrane proteins into a planar bilayer lipid membrane on the surface of a biosensor chip. At the same time, one of the commonly used approaches to working with membrane proteins using various methods is the construction of proteoliposomes containing membrane proteins. In this work, using CYP3A4 and CYB5A as protein partners, we evaluated two approaches to the creation of proteoliposomes: incorporation of a membrane protein into liposomes saturated with detergents and incorporation of a membrane protein into the forming proteoliposomes by the mechanism of micellar coalescence. The interaction of CYP3A4 with proteoliposomes obtained by incorporating CYB5A into detergent-saturated liposomes was shown. On the contrary, interaction between CYP3A4 and proteoliposomes containing CYB5A, obtained by the method of micellar coalescence, was not detected. Thus, it was shown that the incorporation of the membrane protein into liposomes saturated with a detergent was a more preferable method for working with an SPR biosensor as compared to the method of proteoliposomes formation by micellar coalescence. Detailed protocols for the creation of proteoliposomes and SPR-analysis can be useful to a wide range of researchers.

Proteome-wide modulation of S-nitrosylation in Trypanosoma cruzi trypomastigotes upon interaction with the host extracellular matrix

Trypanosoma cruzi trypomastigotes adhere to extracellular matrix (ECM) to invade mammalian host cells regulating intracellular signaling pathways. Herein, resin-assisted enrichment of thiols combined with mass spectrometry were employed to map site-specific S-nitrosylated (SNO) proteins from T. cruzi trypomastigotes incubated (MTy) or not (Ty) with ECM. We confirmed the reduction of S-nitrosylation upon incubation with ECM, associated with a rewiring of the subcellular distribution and intracellular signaling pathways. Forty, 248 and 85 SNO-peptides were identified only in MTy, Ty or in both conditions, respectively. SNO proteins were enriched in ribosome, transport, carbohydrate and lipid metabolisms. Nitrosylation of histones H2B and H3 on Cys64 and Cys126, respectively, is described. Protein-protein interaction networks revealed ribosomal proteins, proteins involved in carbon and fatty acid metabolism to be among the enriched protein complexes. Kinases, phosphatases and enzymes involved in the metabolism of carbohydrates, lipids and amino acids were identified as nitrosylated and phosphorylated, suggesting a post-translational modifications crosstalk. In silico mapping of nitric oxide synthase (NOS) genes, previously uncharacterized, matched to four putative T. cruzi proteins expressing C-terminal NOS domain. Our results provide the first site-specific characterization of S-nitrosylated proteins in T. cruzi and their modulation upon ECM incubation before infection of the mammalian hosts. SignificanceProtein S-nitrosylation represents a major molecular mechanism for signal transduction by nitric oxide. We present for the first time a proteomic profile of S-nitrosylated proteins from infective forms of T. cruzi, showing a decrease in SNO proteins after incubation of the parasite with the extracellular matrix, a necessary step for the parasite invasion of the host mammalian cells. We also show for the first time nitrosylation of H2B (Cys64) and H3 (Cys126) histones, sites not conserved in higher eukaryotic cells, and suggest that some specific histone isoforms are sensitive to NO signaling. S-nitrosylation in H2B and H3 histones are more abundant in MTy. Moreover, proteins involved in translation, glycolytic pathway and fatty acid metabolism are enriched in the present dataset. Comparison of the SNO proteome and the phosphoproteome, obtained previously under the same experimental conditions, show that most of the proteins sharing both modifications are involved in metabolic pathways, transport and ribosome function. The data suggest that both PTMs are involved in reprogramming the metabolism of T. cruzi in response to environmental changes. Although NO synthesis was detected in T. cruzi, the identification of NOS remains elusive. Analysis in silico showed two genes similar in domains to NADPH-dependent cytochrome-P450 reductase and two putative oxidoreductases, but no oxygenase domain of NOS was mapped in the T. cruzi genome. It is tempting to speculate that NO synthase-like from T. cruzi and its early NO-mediated pathways triggered in response to host interaction constitute potential diagnostic and therapeutic targets.

Identification and circumvention of bottlenecks in CYP21A2-mediated premedrol production using recombinant Escherichia coli.

Synthetic glucocorticoids such as methylprednisolone are compounds of fundamental interest to the pharmaceutical industry as their modifications within the sterane scaffold lead to higher inflammatory potency and reduced side effects compared with their parent compound cortisol. In methylprednisolone production, the complex chemical hydroxylation of its precursor medrane in position C21 exhibits poor stereo- and regioselectivity making the process unprofitable and unsustainable. By contrast, the use of a recombinant E. coli system has recently shown high suitability and efficiency. In this study, we aim to overcome limitations in this biotechnological medrane conversion yielding the essential methylprednisolone-precursor premedrol by optimizing the CYP21A2-based whole-cell system on a laboratory scale. We successfully improved the whole-cell process in terms of premedrol production by (a) improving the electron supply to CYP21A2; here we use the N-terminally truncated version of the bovine NADPH-dependent cytochrome P450 reductase (bCPR-27 ) and coexpression of microsomal cytochrome b5 ; (b)enhancing substrate access to the heme by modification of the CYP21A2 substrate access channel; and (c)circumventing substrate inhibition which is presumed to be the main limiting factor of the presented system by developing an improved fed-batch protocol.By overcoming the presented limitations in whole-cell biotransformation, we were able to achieve a more than 100% improvement over the next best system under equal conditions resulting in 691 mg·L-1 ·d-1 premedrol.

A large-scale comparative analysis of affinity, thermodynamics and functional characteristics of interactions of twelve cytochrome P450 isoforms and their redox partners

The aim of the present work was to establish the thermodynamic and functional differences in the protein-protein interactions between the components of the P450-dependent mitochondrial (mit) and microsomal (mic) monooxygenase systems using 12 different isoforms of cytochromes P450 and two redox partners, NADPH-dependent cytochrome P450 reductase (CPR) and adrenodoxin (Adx). Comparative analysis of the affinity, thermodynamics, enzymatic activity and the ability for one-electron reduction has been carried out. The study of protein-protein interactions to determine the equilibrium dissociation constants (Kd) was performed using surface plasmon resonance (SPR) biosensor Biacore 3000. We demonstrated that CPR and Adx interacted with both, micCYPs and mitCYPs, with different affinities (Kd values ranged from 0.01 to 2 μM). All complexes of microsomal (micCYP) and mitochondrial (mitCYP) cytochrome P450 with redox partners can be divided into three groups depending on the prevalent role of either enthalpy or entropy contribution. About 90% of CYP/redox partner complexes were entropy-driven, while the contribution of enthalpy and entropy differed significantly in case of mitCYP/Adx complexes. The CYP11A1/Adx complex was enthalpy-driven, while CYP11B1/Adx and CYP11B2/Adx complexes were entropy-driven. Thermodynamic discrimination of mitCYPs/Adx complexes is likely associated with the different functional impact of CYP11A1 and CYP11B. The exception was the enthalpy-entropy-driven (mixed type) CYP21A2/Adx complex. CPR and Adx were able to transfer the first electron to micCYPs while mitCYPs demonstrated high specificity to Adx. Productive catalysis for mitCYPs observed only in the presence of Adx/AdR pair, while in case of steroidogenic micCYPs (CYP17A1, CYP19A1, and CYP21A2) it was found either in the presence of a CPR or an Adx/AdR pair. From the evolutionary point of view, the type 1 electron transport system (mitCYPs, Adx and NADPH-dependent adrenodoxin reductase (AdR)) increased the specialization of protein-protein interactions (PPI) significantly, which was accompanied by an increase in the specificity of electron transfer. In contrast, the evolution of the type 2 electron transport system (micCYPs and CPR) led to an increase in versatility of PPI as demonstrated for steroidogenic microsomal cytochrome P450s. Our data enhance the current understanding of molecular recognition and summarize qualitative and thermodynamic characteristics of protein-protein interactions in the P450-dependent mitochondrial and microsomal monooxygenase systems.

A Combinatorial Approach To Study Cytochrome P450 Enzymes for De Novo Production of Steviol Glucosides in Baker's Yeast.

Biosynthesis of steviol glycosides in planta proceeds via two cytochrome P450 enzymes (CYPs): kaurene oxidase (KO) and kaurenoic acid hydroxylase (KAH). KO and KAH function in succession with the support of a NADPH-dependent cytochrome P450 reductase (CPR) to convert kaurene to steviol. This work describes a platform for recombinant production of steviol glucosides (SGs) in Saccharomyces cerevisiae, demonstrating the full reconstituted pathway from the simple sugar glucose to the SG precursor steviol. With a focus on optimization of the KO-KAH activities, combinations of functional homologues were tested in batch growth. Among the CYPs, novel KO75 (CYP701) and novel KAH82 (CYP72) outperformed their respective functional homologues from Stevia rebaudiana, SrKO (CYP701A5) and SrKAH (CYP81), in assays where substrate was supplemented to culture broth. With kaurene produced from glucose in the cell, SrCPR1 from S.rebaudiana supported highest turnover for KO-KAH combinations, besting two other CPRs isolated from S.rebaudiana, the Arabidopsis thaliana ATR2, and a new class I CPR12. Some coexpressions of ATR2 with a second CPR were found to diminish KAH activity, showing that coexpression of CPRs can lead to competition for CYPs with possibly adverse effects on catalysis.

Metabolism of ganoderic acids by a Ganoderma lucidum cytochrome P450 and the 3-keto sterol reductase ERG27 from yeast

Ganoderic acids, a group of oxygenated lanostane-type triterpenoids, are the major bioactive compounds produced by the well-known medicinal macro fungus Ganoderma lucidum. More than 150 ganoderic acids have been identified, and the genome of G. lucidum has been sequenced recently. However, the biosynthetic pathways of ganoderic acids have not yet been elucidated. Here, we report the functional characterization of a cytochrome P450 gene CYP512U6 from G. lucidum, which is involved in the ganoderic acid biosynthesis. CYP512U6 hydroxylates the ganoderic acids DM and TR at the C-23 position to produce hainanic acid A and ganoderic acid Jc, respectively. In addition, CYP512U6 can also hydroxylate a modified ganoderic acid DM in which the C-3 ketone has been reduced to hydroxyl by the sterol reductase ERG27 from Saccharomyces cerevisiae. An NADPH-dependent cytochrome P450 reductase from G. lucidum was also isolated and characterized. These results will help elucidate the biosynthetic pathways of ganoderic acids.

Heterologous Co-expression of CYP6B7 and NADPH-Dependent Cytochrome P450 Reductase From Helicoverpa armigera (Lepidoptera: Noctuidae) in Pichia pastoris.

As important metabolic enzymes, the function of cytochrome P450 monooxygenases (CYPs) has been demonstrated repeatedly through various means, including heterologous expression systems. Unfortunately, most model systems typically lack expression of a conspecific NADPH-dependent cytochrome P450 reductase (CPR), which is the electron transfer partner of CYPs. As a result, the activities of heterologously expressed insect CYPs may not accurately reflect detoxification activities in vivo. Previously, CYP6B7 from Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) (HaCYP6B7) has been expressed in the Pichia pastoris GS115 strain and shown to detoxify bifenthrin, fenvalerate and chlorpyrifos. However, it remains to be determined if co-expression of HaCYP6B7 with HaCPR will enhance the detoxification ability of the expression system. In the present study, HaCYP6B7 and HaCPR genes were co-expressed in P. pastoris using a reconstituted expression vector, pPICZA-HaCYP6B7-HaCPR. Protein expression was confirmed by Western blot, and the detoxification activities of microsomal fractions to p-nitroanisole O-demethylation (PNOD), 7-ethoxycoumarin O-deethylation (ECOD), fenvalerate and chlorpyrifos were measured. Co-expression of HaCYP6B7 with HaCPR resulted in PNOD and ECOD activities of 1.90 nmol/min/mg·protein and 12.39 pmol/min/mg·protein, which were 1.6- and 1.5-fold of that catalyzed by HaCYP6B7 expressed alone, respectively. Furthermore, microsomes of pPICZA-HaCYP6B7-HaCPR-GS115 had higher detoxification activity than that of pPICZA-HaCYP6B7-GS115 to fenvalerate, but not chlorpyrifos. The results indicated that co-expression of HaCYP6B7 with conspecific CPR could enhance the detoxification activities to some substrates comparing with expression of HaCYP6B7 alone.

Effect of genetic polymorphism of human CYP2B6 on the metabolic activation of chlorpyrifos

Chlorpyrifos (CPS) is a broad-spectrum organophosphate insecticide that is neurotoxic in humans. Chlorpyrifos oxon (CPO) is a toxic metabolite of CPS that is produced by CYP2B6. In this study, we examined the variability of CPS metabolism resulting from single-nucleotide polymorphisms in CYP2B6. Wild-type CYP2B6 (CYP2B6.1) and two variants each with a single amino acid substitution: CYP2B6.5 (R487C) and CYP2B6.8 (K139E) were co-expressed together with human NADPH-dependent cytochrome P450 reductase in Escherichia coli (E. coli). Both of the CYP2B6 variants were successfully expressed in E. coli. The conversion of CPS to CPO by the CYP2B6 variants was analyzed with high-performance liquid chromatography. Km and Vmax of the reaction by CYP2B6.1 were 18.50±2.94μM and 17.07±1.15mol/min/mol P450, respectively. The CYP2B6 variants produced CPO with the following kinetic parameters: Km for CYP2B6.5 and CYP2B6.8 were 20.44±6.43 and 44.69±9.97μM, respectively; and Vmax were 1.10±0.10 and 1.77±0.26mol/min/mol P450, respectively. These results indicate that the amino acid substitutions in the CYP2B6 variants suppressed the metabolic activation of CPS. CYP2B6 variants have altered capacity to bioactivate CPF and may affect individual susceptibility of CPF.