P450 Reductase Research Articles

Plant species compositions alleviate toxicological effects of bisphenol A by enhancing growth, antioxidant defense system, and detoxification

Bisphenol A (BPA), a broadly disseminated endocrine disturbing chemicals in environment, is harmful to creatures and plants. Plants can uptake and metabolize BPA, but a single plant species ability is limited. Undeniably, plant species compositions have a more vital ability to remove pollutants than a single plant species. However, the mechanisms of plant species compositions alleviating toxicological effects of bisphenol A are poorly understood. Here, we administered plant species compositions, which based on a full-factorial design of Phragmites australis (A), Typha latifolia (B), and Arundo donax (C), to unveil their role in BPA exposure. The results illustrated that the root activity, biomass, and photosynthetic pigment contents of the mixed hydroponic group (e.g., sp(ABC)) were significantly increased under concentration of BPA(1.5, 5, and 10mg L-1), which showed that the root activity, fresh weight, dry weight, chlorophyll a, and total chlorophyll contents of shoots were increased. While mixed-hydroponic culture groups (e.g., sp(AB), sp(ABC)) significantly increased antioxidant enzyme activity and antioxidant substances under concentration of BPA(5 and 10mg L-1), it astoundingly diminished responsive oxygen species (ROS) and malondialdehyde (MDA) substance, proposing that mixed-hydroponic culture groups calmed oxidative stress. Further analysis revealed that mixed-hydroponic culture groups (e.g., sp(AB), sp(AC), sp(ABC)) of 1.5, 5, and 10mg L-1 BPA exposure significantly increased detoxification enzyme activity of NADPH-cytochrome P450 reductase (CPR), glutathione S-transferase (GST), and glycosyltransferase (GT). Moreover, mixed-hydroponic culture groups (e.g., sp(AB), sp(AC), sp(ABC)) decreased the BPA substance in leaves, proposing that mixed-hydroponic culture groups advanced BPA metabolism by improving CPR, GST, and GT enzyme activities. These results demonstrated that a mixed-hydroponic culture strategy can alleviate BPA phytotoxicity and possibly offer natural and potential phytoremediation methods for BPA.

Biochemical Investigation of Membrane-Bound Cytochrome b5 and the Catalytic Domain of Cytochrome b5 Reductase from Arabidopsis thaliana.

The endoplasmic reticulum (ER) membrane of plant cells contains several enzymes responsible for the biosynthesis of a diverse range of molecules essential for plant growth and holds potential for industrial applications. Many of these enzymes are dependent on electron transfer proteins to sustain their catalytic cycles. In plants, two crucial ER-bound electron transfer proteins are cytochrome b5 and cytochrome b5 reductase, which catalyze the stepwise transfer of electrons from NADH to redox enzymes such as fatty acid desaturases, cytochrome P450s, and plant aldehyde decarbonylase. Despite the high significance of plant cytochrome b5 and cytochrome b5 reductase, they have eluded detailed characterization to date. Here, we overexpressed the full-length membrane-bound cytochrome b5 isoform B from the model plant Arabidopsis thaliana in Escherichia coli, purified the protein employing detergents as well as styrene-maleic acid (SMA) copolymers, and biochemically characterized the protein. The SMA-encapsulated cytochrome b5 exhibits a discoidal shape and the characteristic features of the active heme-bound state. We also overexpressed and purified the soluble domain of cytochrome b5 reductase from A. thaliana, establishing its activity, stability, and kinetic parameters. Further, we demonstrated that the plant cytochrome b5, purified in detergents and styrene maleic acid lipid particles (SMALPs), readily accepts electrons from the cognate plant cytochrome b5 reductase and distant electron mediators such as plant NADPH-cytochrome P450 oxidoreductase and cyanobacterial NADPH-ferredoxin reductase. We also measured the kinetic parameters of cytochrome b5 reductase for cytochrome b5. Our studies are the first to report the purification and detailed biochemical characterization of the plant cytochrome b5 and cytochrome b5 reductase from the bacterial overexpression system.

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.

Evaluation of biotransformation capacity of transplastomic plants and hairy roots of Nicotiana tabacum expressing human cytochrome P450 2D6

Cytochrome P450 monooxygenases (CYPs) are important tools for regio- and stereoselective oxidation of target molecules or engineering of metabolic pathways. Functional heterologous expression of eukaryotic CYPs is often problematic due to their dependency on the specific redox partner and the necessity of correct association with the membranes for displaying enzymatic activity. Plant hosts offer advantages of accessibility of reducing partners and a choice of membranes to insert heterologous CYPs. For the evaluation of plant systems for efficient CYP expression, we established transplastomic plants and hairy root cultures of Nicotiana tabacum carrying the gene encoding human CYP2D6 with broad substrate specificity. The levels of CYP2D6 transcript accumulation and enzymatic activity were estimated and compared with the data of CYP2D6 transient expression in N. benthamiana. The relative level of CYP2D6 transcripts in transplastomic plants was 2–3 orders of magnitude higher of that observed after constitutive or transient expression from the nucleus. CYP2D6 expressed in chloroplasts converted exogenous synthetic substrate loratadine without the need for co-expression of the cognate CYP reductase. The loratadine conversion rate in transplastomic plants was comparable to that in N. benthamiana plants transiently expressing a chloroplast targeted CYP2D6 from the nucleus, but was lower than the value reported for transiently expressed CYP2D6 with the native endoplasmic reticulum signal-anchor sequence. Hairy roots showed the lowest substrate conversion rate, but demonstrated the ability to release the product into the culture medium. The obtained results illustrate the potential of plant-based expression systems for exploiting the enzymatic activities of eukaryotic CYPs with broad substrate specificities.

Transcriptome Analysis and Identification of the Cholesterol Side Chain Cleavage Enzyme BbgCYP11A1 From Bufo bufo gargarizans.

Bufo bufo gargarizans Cantor are precious medicinal animals in traditional Chinese medicine (TCM). Bufadienolides as the major pharmacological components are generated from the venomous glands of B. bufo gargarizans. Bufadienolides are one type of cardiac aglycone with a six-member lactone ring and have properties of antitumor, cardiotonic, tonsillitis, and anti-inflammatory. The biosynthesis of bufadienolides is complex and unclear. This study explored the transcriptome of three different tissues (skin glands, venom glands, and muscles) of B. bufo gargarizans by high-throughput sequencing. According to the gene tissue–specific expression profile, 389 candidate genes were predicted possibly participating in the bufadienolides biosynthesis pathway. Then, BbgCYP11A1 was identified as a cholesterol side chain cleaving the enzyme in engineering yeast producing cholesterol. Furthermore, the catalytic activity of BbgCYP11A1 was studied with various redox partners. Interestingly, a plant NADPH-cytochrome P450 reductase (CPR) from Anemarrhena asphodeloides showed notably higher production than BbgAdx-2A-BbgAdR from B. bufo gargarizans. These results will provide certainly molecular research to reveal the bufadienolides biosynthesis pathway in B. bufo gargarizans.

Loss of Endothelial Cytochrome P450 Reductase Induces Vascular Dysfunction in Mice.

POR (cytochrome P450 reductase) provides electrons for the catalytic activity of the CYP (cytochrome P450) monooxygenases. CYPs are dual-function enzymes as they generate protective vasoactive mediators derived from polyunsaturated fatty acids but also reactive oxygen species. It is not known in which conditions the endothelial POR/CYP system is beneficial versus deleterious. Here, the activity of all CYP enzymes was eliminated in the vascular endothelium to examine its impact on vascular function. An endothelial-specific, tamoxifen-inducible POR knockout mouse (ecPOR-/-) was generated. Vascular function was studied by organ chamber experiments. eNOS (endothelial nitric oxide synthase) activity was accessed by heavy arginine/citrulline LC-MS/MS detection and phosphorylation of serine1177 in aortic rings. CYP-derived epoxyeicosatrienoic acids and prostanoids were measured by LC-MS/MS. Gene expression of aorta and endothelial cells was profiled by RNA sequencing. Blood pressure was measured by telemetry. Acetylcholine-induced endothelium-dependent relaxation was attenuated in isolated vessels of ecPOR-/- as compared with control mice. Additionally, ecPOR-/- mice had attenuated eNOS activity and eNOS/AKT phosphorylation. POR deletion reduced endothelial stores of CYP-derived epoxyeicosatrienoic acids but increased vascular prostanoids. This phenomenon was paralleled by the induction of genes implicated in eicosanoid generation. In response to Ang II (angiotensin II) infusion, blood pressure increased significantly more in ecPOR-/- mice. Importantly, the cyclooxygenase inhibitor Naproxen selectively lowered the Ang II-induced hypertension in ecPOR-/- mice. POR expression in endothelial cells maintains eNOS activity and its loss results in an overactivation of the vasoconstrictor prostanoid system. Through these mechanisms, loss of endothelial POR induces vascular dysfunction and hypertension.

Conformational Rearrangements in the Redox Cycling of NADPH-Cytochrome P450 Reductase from Sorghum bicolor Explored with FRET and Pressure-Perturbation Spectroscopy.

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

Cytochrome P450 Monooxygenase/Cytochrome P450 Reductase Bi-Enzymatic System Isolated From Ilex asprella for Regio-Specific Oxidation of Pentacyclic Triterpenoids.

Ilex asprella is a plant from Aquifoliaceae. Its root is commonly used as folk medicinal materials in southern China. The chemical compositions of I. asprella are rich in pentacyclic triterpenoids, which show various biological activities and demonstrate a good prospect for drug development. The elucidation of biosynthesis mechanism of triterpenoids in I. asprella could lay important foundations for the production of these precious plant secondary metabolites by metabolic engineering. Our previous studies have revealed IaAO1 (a CYP716A210 homolog) responsible for the C-28 oxidation of α- and β-amyrin. Herein, we reported the identification of three more cytochrome P450 monooxygenase genes IaAO2 (a CYP716A212 homolog), IaAO4 (CYP714E88), IaAO5 (CYP93A220), and a cytochrome P450 reductase gene IaCPR by using Saccharomyces cerevisiae eukaryotic expression system and gas chromatography-mass spectrometry. Among them, the protein encoded by IaAO2 can catalyze the C-28 oxidation of α-amyrin and β-amyrin, IaAO4 can catalyze the C-23 oxidation of ursolic acid and oleanolic acid, while IaAO5 is responsible for the C-24 oxidation of β-amyrin. By introducing three genes IaAO1, IaAO4 and IaCPR into S. cerevisiae. We constructed an engineered yeast strain that can produce C-23 hydroxyl ursane-type triterpenoid derivatives. This study contributes to a thorough understanding of triterpenoid biosynthesis of medicinal plants and provides important tools for further metabolic engineering.

Antioxidant potential of biotransformed green tea catechin metabolites and their impact on peripheral blood mononuclear cells

ObjectiveCatechin absorption, bioavailability, and distribution in human tissue and excretion were low. Colonic bacteria degrade catechin and few studies suggest that biotransformed metabolites may increase or decrease biological activities. However, the antioxidant potential of biotransformed metabolites is still unexplored. Hence, it is necessary to investigate the antioxidant potential of colonic bacterial transformed catechin metabolites. MethodsThe In vitro antioxidant potential of biotransformed catechin metabolites was assessed by DPPH, Hydrogen peroxide, and ABTS assay. Cytotoxic effect of biotransformed catechin metabolites on human mesenchymal stem cells and their response to antioxidant genes GPX1 and POR expression in human peripheral blood mononuclear cells (PBMC) were evaluated. ResultsFree radical scavenging activity of partially purified bacterial transformed catechin metabolites was low compared to pure catechin. The results revealed that variations in the antioxidant potential of pure catechin and colonic bacterial altered catechin metabolites. The IC50 value of partially purified biotransformed catechin metabolites exhibits free radical scavenging activity at high concentrations, while pure catechins act as a potent inhibitor at lower concentrations. The expression of GPX1 and POR gene increased four folds in a concentration dependent manner in pure catechin-treated cells. In contrast, partially purified biotransformed catechin metabolites treated cells expressed the downregulation of GPX1 and upregulation of POR gene. ConclusionTherefore, it can be concluded that the functional group alteration in biotransformed catechin metabolites may reduce the hydrogen donating ability, thus In vitro antioxidant potential of biotransformed catechin metabolites was low compared to pure catechin. Hence, GPX1 and POR exhibit different expression patterns in treated peripheral blood mononuclear cells. These findings suggest that pure catechin and colonic bacterial transformed catechin metabolites differed functionally.

Classic and current concepts in adrenal steroidogenesis: a reappraisal.

Adrenal steroid biosynthesis and its related pathology are constant evolving disciplines. In this paper, we review classic and current concepts of adrenal steroidogenesis, plus control mechanisms of steroid pathways, distribution of unique enzymes and cofactors, and major steroid families. We highlight the presence of a "mineralocorticoid (MC) pathway of zona fasciculata (ZF)", where most circulating corticosterone and deoxycorticosterone (DOC) originate together with 18OHDOC, under ACTH control, a claim based on functional studies in normal subjects and in patients with 11β-, and 17α-hydroxylase deficiencies. We emphasize key differences between CYP11B1 (11β-hydroxylase) and CYP11B2 (aldosterone synthase) and the onset of a hybrid enzyme - CYP11B1/CYP11B2 -, responsible for aldosterone formation in ZF under ACTH control, in "type I familial hyperaldosteronism" (dexamethasone suppressible). In "apparent MC excess syndrome", peripheral conversion of cortisol to cortisone is impaired by lack of 11β-hydroxysteroid dehydrogenase type 2, permitting free cortisol access to MC receptors resulting in severe hypertension. We discuss two novel conditions involving the synthesis of adrenal androgens: the "backdoor pathway", through which dihydrotestosterone is formed directly from androsterone, being relevant for the fetoplacental setting and sexual differentiation of male fetuses, and the rediscovery of C19 11-oxygenated steroids (11-hydroxyandrostenedione and 11-ketotestosterone), active androgens and important markers of virilization in 21-hydroxylase deficiency and polycystic ovaries syndrome. Finally, we underline two enzyme cofactor deficiencies: cytochrome P450 oxidoreductase which partially affects 21- and 17α-hydroxylation, producing a combined clinical/hormonal picture and causing typical skeletal malformations (Antley-Bixler syndrome), and PAPSS2, coupled to SULT2A1, that promotes sulfation of DHEA to DHEAS, preventing active androgens to accumulate. Its deficiency results in reduced DHEAS and elevated DHEA and androgens with virilization. Future and necessary studies will shed light on remaining issues and questions on adrenal steroidogenesis.

Biodegradation of tricresyl phosphates isomers by a novel microbial consortium and the toxicity evaluation of its major products

A novel microbial consortium ZY1 capable of degrading tricresyl phosphates (TCPs) was isolated, it could quickly degrade 100% of 1 mg/L tri-o-cresyl phosphate (ToCP), tri-p-cresyl phosphate (TpCP) and tri-m-cresyl phosphate (TmCP) within 36, 24 and 12 h separately and intracellular enzymes occupied the dominated role in TCPs biodegradation. Additionally, triphenyl phosphate (TPHP), 2-ethylhexyl diphenyl phosphate (EHDPP), bisphenol-A bis (diphenyl phosphate) (BDP), tris (2-chloroethyl) phosphate (TCEP) and tris (1-chloro-2-propyl) phosphate (TCPP) could also be degraded by ZY1 and the aryl-phosphates was easier to be degraded. The TCPs reduction observed in freshwater and seawater indicated that high salinity might weak the degradability of ZY1. The detected degradation products suggested that TCPs was mainly metabolized though the hydrolysis and hydroxylation. Sequencing analysis presented that the degradation of TCPs relied on the cooperation between sphingobacterium, variovorax and flavobacterium. The cytochrome P450/NADPH-cytochrome P450 reductase and phosphatase were speculated might involve in TCPs degradation. Finally, toxicity evaluation study found that the toxicity of the diesters products was lower than their parent compound based on the generation of the intracellular reactive oxygen (ROS) and the apoptosis rate of A549 cell. Taken together, this research provided a new insight for the bioremediation of TCPs in actual environment.

CPR Gene Contributes to Integument Function and Ovary Development in a Rice Planthopper.

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

Hydroxycinnamoyltransferase and CYP98 in phenolic metabolism in the rosmarinic acid-producing hornwort Anthoceros agrestis

Main conclusionAnthoceros agrestis hydroxycinnamoyltransferase accepts shikimic and 3-hydroxyanthranilic acids while hydroxycinnamoylester/amide 3-hydroxylase (CYP98A147) preferred p-coumaroyl-(3-hydroxy)anthranilic acid compared to the shikimic acid derivative. Alternative pathways towards rosmarinic acid have to be considered.Rosmarinic acid (RA) is a well-known ester of caffeic acid and 3,4-dihydroxyphenyllactic acid. In the search for enzymes involved in RA biosynthesis in the hornwort Anthoceros agrestis, the hydroxycinnamoyltransferase sequence with the highest similarity to rosmarinic acid synthase from Lamiaceae has been amplified and heterologously expressed in Escherichia coli. In parallel, the single cytochrome P450 sequence belonging to the CYP98 group in Anthoceros agrestis was isolated and expressed in Saccharomyces cerevisiae which did not result in protein formation. Codon optimization and co-expression with NADPH:cytochrome P450 reductase (CPR) from Coleus blumei resulted in the formation of active enzymes. Both, the hydroxycinnamoyltransferase and CYP98 were characterized with respect to their temperature and pH optimum as well as their substrate acceptance. The hydroxycinnamoyltransferase (AaHCT6) readily accepted p-coumaroyl- and caffeoyl-CoA with a slightly higher affinity towards p-coumaroyl-CoA. The best acceptor substrate was shikimic acid (Km 25 µM with p-coumaroyl-CoA) followed by 3-hydroxyanthranilic acid (Km 153 µM with p-coumaroyl-CoA). Another accepted substrate was 2,3-dihydroxybenzoic acid. Anthranilic acid and 4-hydroxyphenyllactic acid (as precursor for RA) were not used as substrates. p-Coumaroylesters and -amides are substrates hydroxylated by CYP98 hydroxylases. The only CYP98 sequence from Anthoceros agrestis is CYP98A147. The best substrates for the NADPH-dependent hydroxylation were p-coumaroylanthranilic and p-coumaroyl-3-hydroxyanthranilic acids while p-coumaroylshikimic and p-coumaroyl-4-hydroxyphenyllactic acids were poor substrates. The biosynthetic pathway towards rosmarinic acid thus still remains open and other enzyme classes as well as an earlier introduction of the 3-hydroxyl group to afford the caffeic acid substitution pattern must be taken into consideration.

Bromodomain containing 4 transcriptionally activated Deltex E3 ubiquitin ligase 2 contributes to glioma progression and predicts an unfavorable prognosis.

BackgroundGlioblastoma multiforme (GBM) is the most common type of glioma, and the most aggressive brain malignancy in adults. This study sought to identify novel survival-status related markers, and examine their function in glioma.MethodsThe gene expression, survival heatmaps, and Kaplan-Meier survival plots of the genes were analyzed by using gene expression profiling interactive analysis (GEPIA) dataset, Linked Omics. The single-cell data analysis and tumor immune infiltration analysis was conducted by Tumor Immune Estimation Resource (TIMER) dataset. DBTRG and U251 cells with silenced Deltex E3 ubiquitin ligase 2 (DTX2) expression were constructed and used for Cell Counting Kit 8 (CCK-8), and wound healing assay in vitro. Chromatin immunoprecipitation sequencing (ChIP-seq) analysis was used to explore the histone activation marks and transcription factors DTX2 promoter. Dual-luciferase assays were carried out to detect the luciferase activities of bromodomain containing 4 (BRD4) binding to DTX2.ResultsWe first conducted a survival-status analysis to identify survival status-related genes in The Cancer Genome Atlas GBM and low-grade glioma data sets. A subsequent analysis identified 3 novel prognostic biomarkers; that is, DTX2, cytochrome P450 oxidoreductase, and Williams-Beuren syndrome chromosomal region 16 protein. In the validation Chinese Glioma Genome Atlas data sets, DTX2 showed the best performance, and was examined in a further analysis. Next, 3 short-hairpin ribonucleic acids were designed to silence DTX2 expression, and CCK-8 and wound-healing assays were applied to study the function of DTX2. We found that DTX2-silenced glioma cells exhibited a significant decrease in their growth and migration capabilities. Finally, the molecular basis for increased DTX2 in glioma was investigated via ChIP-Seq analysis and luciferase assays. The analysis revealed that DTX2 was transcriptionally activated by BRD4.ConclusionsIn conclusion, BRD4 transcriptionally activates DTX2, contributes to glioma progression, predicts an unfavorable prognosis, and could provide new options for glioma prognosis prediction and treatment.

Developing Multi-Copy Chromosomal Integration Strategies for Heterologous Biosynthesis of Caffeic Acid in Saccharomyces cerevisiae.

Caffeic acid, a plant-sourced phenolic compound, has a variety of biological activities, such as antioxidant and antimicrobial properties. The caffeic acid biosynthetic pathway was initially constructed in S. cerevisiae, using codon-optimized TAL (coTAL, encoding tyrosine ammonia lyase) from Rhodobacter capsulatus, coC3H (encoding p-coumaric acid 3-hydroxylase) and coCPR1 (encoding cytochrome P450 reductase 1) from Arabidopsis thaliana in 2 μ multi-copy plasmids to produce caffeic acid from glucose. Then, integrated expression of coTAL via delta integration with the POT1 gene (encoding triose phosphate isomerase) as selection marker and episomal expression of coC3H, coCPR1 using the episomal plasmid pLC-c3 were combined, and caffeic acid production was proved to be improved. Next, the delta and rDNA multi-copy integration methods were applied to integrate the genes coC3H and coCPR1 into the chromosome of high p-coumaric acid yielding strain QT3-20. The strain D9 constructed via delta integration outperformed the other strains, leading to 50-fold increased caffeic acid production in optimized rich media compared with the initial construct. The intercomparison between three alternative multi-copy strategies for de novo synthesis of caffeic acid in S. cerevisiae suggested that delta-integration was effective in improving caffeic acid productivity, providing a promising strategy for the production of valuable bio-based chemicals in recombinant S. cerevisiae.

Impact of polymorphisms of pharmacokinetics-related genes and the inflammatory response on the metabolism of voriconazole.

The effects of inflammatory responses and polymorphisms of the genes encoding cytochrome P450 (CYP) (CYP2C19 and CYP3A5), flavin‐containing monooxygenase 3 (FMO3), pregnane X receptor (NR1I2), constitutive androstane receptor (NR1I3), and CYP oxidoreductase (POR) on the ratio of voriconazole (VRCZ) N‐oxide to VRCZ (VNO/VRCZ) and steady‐state trough concentrations (C0h) of VRCZ were investigated. A total of 56 blood samples were collected from 36 Japanese patients. Results of multiple linear regression analyses demonstrated that the presence of the extensive metabolizer CYP2C19 genotype, the dose per administration, and the presence of the NR1I2 rs3814057 C/C genotype were independent factors influencing the VNO/VRCZ ratio in patients with CRP levels of less than 40 mg/L (standardized regression coefficients (SRC) = 0.448, −0.301, and 0.390, respectively; all p < .05). With regard to the concentration of VRCZ itself, in addition to the above factors, the presence of the NR1I2 rs7643645 G/G and rs3814055 T/T genotypes were found to be independent factors influencing the VRCZ C0h in these patients (SRC = −0.430, 0.424, −0.326, 0.406 and −0.455, respectively; all p < .05). On the contrary, in patients with CRP levels of at least 40 mg/L, no independent factors were found to affect VNO/VRCZ and VRCZ C0h. Inflammatory responses, and CYP2C19 and NR1I2 polymorphisms may be useful information for the individualization of VRCZ dosages.

Use of a Baculovirus-Mammalian Cell Expression-System for Expression of Drug-Metabolizing Enzymes: Optimization of Infection With a Focus on Cytochrome P450 3A4.

Heterologous expression systems are important for analyzing the effects of genetic factors including single nucleotide polymorphisms on the functions of drug-metabolizing enzymes. In this study, we focused on a baculovirus-mammalian cell (Bac-Mam) expression system as a safer and more efficient approach for this purpose. The baculovirus-insect cell expression system is widely utilized in large-scale protein expression. Baculovirus has been shown to also infect certain mammalian cells, although the virus only replicates in insect cells. With this knowledge, baculovirus is now being applied in a mammalian expression system called the Bac-Mam system wherein a gene-modified baculovirus is used whose promotor is replaced with one that can function in mammalian cells. We subcloned open-reading frames of cytochrome P450 3A4 (CYP3A4), UDP-glucuronosyltransferase (UGT) 1A1, and UGT2B7 into a transfer plasmid for the Bac-Mam system, and prepared recombinant Bac-Mam virus. The obtained virus was amplified in insect Sf9 cells and used to infect mammalian COS-1 cells. Expression of CYP3A4, UGT1A1, and UGT2B7 in COS-1 cell homogenates were confirmed by immunoblotting. Optimum infection conditions including the amount of Bac-Mam virus, culture days before collection, and concentration of sodium butyrate, an enhancer of viral-transduction were determined by monitoring CYP3A4 expression. Expressed CYP3A4 showed appropriate activity without supplying hemin/5-aminolevulinic acid or co-expressing with NADPH-cytochrome P450 reductase. Further, we compared gene transfer efficiency between the Bac-Mam system and an established method using recombinant plasmid and transfection reagent. Our results indicate that the Bac-Mam system can be applied to introduce drug-metabolizing enzyme genes into mammalian cells that are widely used in drug metabolism research. The expressed enzymes are expected to undergo appropriate post-translational modification as they are in mammalian bodies. The Bac-Mam system may thus accelerate pharmacogenetics and pharmacogenomics research.

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.

Production of hesperetin from naringenin in an engineered Escherichia coli consortium

Hesperetin, a methoxylated flavanone, has numerous biological activities. Access to this compound is currently restricted by its low abundance in plants, which limits its practical applicability. To provide an alternative, eco-friendly production source, we developed a biosynthetic pathway of hesperetin in an engineered Escherichia coli consortium, which was fed with naringenin as a precursor and demonstrated good hesperetin production. The biosynthetic pathway was divided into two modules. The first recombinant host harbored the pathway genes from two different species: a flavonoid 3′-hydroxylase (F3'H) gene from Gentiana triflora and a cytochrome P450 reductase (CPR) gene from Arabidopsis thaliana. The second strain heterologously expressed a gene encoding a flavonoid 4′-O-methyltransferase (MpOMT) from Mentha × piperita, which was N-terminally fused to a Sumo tag. A construct expressing a 29 aa N-terminally truncated F3'H and CPR was the most effective combination for the conversion of naringenin. The strain expressing the Sumo-tagged MpOMT protein exhibited an increase in the final hesperetin titer, reaching 5.9 mg/L. Simultaneous overexpression of metK (coding for the endogenous S-adenosyl-l-methionine [SAM] synthase) further improved the hesperetin titer by 25.1%. Finally, the designed E. coli consortium harboring the two modules efficiently converted naringenin to hesperetin (37.1 mg/L). This work reports the construction of a multi-step in vivo cascade biocatalyst for the biotransformation of naringenin to hesperetin. It also illustrates the potential of the E. coli consortium system for producing other O-methylated flavonoids.

Thermodynamic Driving Forces of Redox-Dependent CPR Insertion into Biomimetic Endoplasmic Reticulum Membranes.

Cytochrome P450 reductase (CPR) is a NADPH-dependent membrane-bound oxidoreductase found in the endoplasmic reticulum (ER) and is the main redox partner for most cytochrome P450 enzymes. Presented are the measured thermodynamic driving forces responsible for how strongly CPR partitions into a biomimetic ER with the same lipid composition of a natural ER. Using temperature-dependent fluorescence correlation spectroscopy and fluorescence single-protein tracking, the standard state free energies, enthalpies, and entropies of the CPR insertion process were all measured. The results of this study demonstrate that the thermodynamic driving forces are dependent on the redox states of CPR. In particular, the partitioning of CPRox into a biomimetic ER is an exothermic process with a small positive change in entropy, while CPRred partitioning is endothermic with a large positive change in entropy. Both resulted in negative free energies and strong association to the biomimetic ER, but the KP of CPRox insertion is measurably smaller than that of CPRred. Using this new information and known results from literature sources, we also present a phenomenological model that accounts for membrane-protein interactions, protein orientation relative to the membrane, and protein conformation as a function of the redox state.