NADPH-P450 Reductase Research Articles

Involvement of P450s in the metabolic resistance of Digitaria sanguinalis (L.) Scop. To ALS-inhibiting herbicides

Weed resistance to a range of herbicides has rapidly evolved, often with different mechanisms of action. The resulting uninhibited growth of weeds poses demonstrable threats to crop production and sustainable agriculture. Digitaria sanguinalis (L.) Scop., a troublesome weed in corn and other agricultural fields, has developed resistance to herbicides that inhibiting ALS (Acetolactate Synthase), such as nicosulfuron. Understanding the weed's resistance patterns and mechanisms is crucial. However, little is known of the non-target site resistance (NTSR) mechanisms of D. sanguinalis owing to a lack of relevant genome sequences and other materials. Therefore, in this study, a population of D.sanguinalis presenting multiple resistance was tested and found that its high level of resistance to ALS-inhibiting herbicides was not associated with target-related alterations.Administration of P450 inhibitors reversed the resistance to ALS-inhibiting herbicides. Following the application of ALS-inhibiting herbicides, the activities of NADPH-P450 reductase and p-nitroanisole O-demethylase (PNOD) were notably greater in the resistant population of D. sanguinalis than those in the susceptible population. The results suggested P450 enzyme familyplays a major role in the metabolic resistance mechanism, that increased P450 enzyme activity promote cross-resistance in D. sanguinalis to ALS-inhibiting herbicides. RNA-seq analysis showed that five genes from the P450 family (CYP709B2, CYP714C2, CYP71A1, CYP76C2, and CYP81E8) were upregulated in resistant D. sanguinalis. In conclusion, the upregulation of several P450 genes is responsible for establishing resistance to ALS-inhibiting herbicides in D. sanguinalis.

CYP102A1 peroxygenase catalyzed reaction via in situ H2O2 generation

CYP102A1 is a promiscuous bacterial cytochrome P450 (CYP or P450) known for its diverse substrates and comparable activity with human P450 enzymes. The development of CYP102A1 peroxygenase activity can contribute significantly to human drug development and drug metabolite production. Peroxygenase has recently emerged as an alternative to a dependency of P450 on NADPH-P450 reductase and NADPH cofactor and gives more opportunity for practical application. However, the H2O2 dependency also leads to challenges regarding its practical application, in which the excessive H2O2 concentration causes the activation of the peroxygenases. Therefore, we need the optimization of H2O2 production to minimize oxidative inactivation. In this study, we report the CYP102A1 peroxygenase-catalyzed atorvastatin hydroxylation reaction with an enzymatic H2O2 generation using glucose oxidase. Random mutagenesis at the CYP102A1 heme domain was used to generate mutant libraries with high throughput screening of highly active mutants, which can pair with the in situ H2O2 generation. The setup of the CYP102A1 peroxygenase reaction was also possible for other statin drugs and could be developed to produce drug metabolites. We also found a relationship between enzyme inactivation and product formation during the catalytic reaction, supported by enzymatic in situ H2O2 supply. It can be suggested that the low product formation is due to enzyme inactivation.

Salinity variation modulates cellular stress response to ZnO nanoparticles in a sentinel marine bivalve, the blue mussel Mytilussp.

Zinc oxide nanoparticles are released into marine environments from industrial, medical and consumer uses sparking concerns about their potential ecotoxicological effects. Ecological hazard assessment of nZnO in marine ecosystems is hindered by the lack of understanding of the potential interactive effects of nZnO toxicity with other common abiotic stressors, such as salinity fluctuations, in marine organisms. To close this gap in our knowledge, we carried out a comprehensive biomarker-based assessment of the combined effects of salinity and nZnO in a sentinel marine bivalve, the blue mussels Mytilus edulis. The mussels were exposed for 21 days to clean seawater (control), an environmentally relevant concentration (100 μg Zn l−1) of nZnO or dissolved Zn (to identify the toxic effects attributable to Zn2+ toxicity) under the normal (15), low (5) and fluctuating (5–15) salinity regimes. The selected molecular and biochemical markers focused on the oxidative stress, apoptosis, detoxification system and inflammation in the gills and the digestive gland of the mussels. Biomarker analysis showed different effects of nZnO and dissolved Zn on biomarkers of oxidative stress, xenobiotic detoxification and apoptosis but similar effects of both pollutants on the levels of metallothioneins and inflammatory markers. Exposure to nZnO led to elevated levels of lipid peroxidation, upregulation of p53 and p38 stress kinases and apoptosis-related genes, most notably in the gills. Exposure to dissolved Zn led to accumulation of protein carbonyls and activated redox-sensitive detoxification enzymes (NADPH-P450 reductase and glutathione-S-transferase) in the mussels. The ambient salinity had significant effects the cellular adverse effects of nZnO in the mussels. The nZnO-induced cellular stress was detectable under the normal (15) and fluctuating (5–15) salinity conditions in the studied brackish water population of the mussels. At low salinity (5), nZnO toxicity signal was almost completely dampened. These findings indicate that chronic osmotic stress close to the tolerance limits of M. edulis prevails over the effects of the environmentally relevant nZnO and dissolved Zn concentrations in combined exposures. These stressor interactions might ameliorate the cellular toxicity of nZnO in the mussels but limit applicability of cellular stress biomarkers for detecting the toxic effects of nanopollutants in low salinity habitats.

The Therapeutic Potential of 4-Methoxy-1-methyl-2-oxopyridine-3-carbamide (MMOXC) Derived from Ricinine on Macrophage Cell Lines Infected with Methicillin-Resistant Strains of Staphylococcus aureus.

The incidences of methicillin-resistant strains of Staphylococcus aureus (MRSA) and their survival inside the macrophages are the major attributes of the relapsed infections after antimicrobial therapy, and it is a global problem. In this context, we have previously demonstrated 4-methoxy-1-methyl-2-oxopyridine-3-carbamide (MMOXC), a Ricinine derivative exhibiting anti-S. aureus and anti-biofilm characteristics by competitively inhibiting uridine monophosphate kinase (UMPK), UDP-N-acetyl muramyl pentapeptide ligase (Mur-F), and peptidyl deformylase, (PDF). In the present study, the stability of this competitive inhibitor MMOXC was evaluated by showing its ability to remain bound to the active sites of UMPK, Mur-F, and PDF even after increasing the incubation time, temperature, pH, and substrate concentration. On growing MRSA in fewer concentrations of MMOXC, these strains could not attain resistance to MMOXC and at the same time distinct reductions in the expression of UMPK, Mur-F, and PDF genes were noted. In vitro, infective models were generated by infecting MRSA to RAW 264.7 and human monocyte-derived macrophage (hMDM) cell lines. In these infected cell lines, in spite of increased nitric oxide synthase (NOS), NADPH-P450 reductase, superoxide dismutase, catalase, and peroxidase activities, the MRSA survived. At 640µM/ml, the concentration of MMOXC penetrated into these infected cells and obliterated MRSA. While treating uninfected macrophage cell lines with MMOXC, no appreciable effect was observed indicating that MMOXC is the most suitable drug for the treatment of infections caused by MRSA.

Resistance of Sogatella furcifera to triflumezopyrim mediated with the overexpression of CYPSF01 which was regulated by nuclear receptor USP

Sogatella furcifera is one of the main agricultural pests in many Asian countries, bringing about enormous injury. A triflumezopyrim-resistant (Tri) strain of S. furcifera was established through continuous screening in laboratory. The determination of synergist and enzyme activity indicated that P450s, especially for the upregulation expression of CYPSF01, played a key role in the increased resistance, confirmed by RNAi, and the recombinant protein of CYPSF01 and NADPH-P450 reductase was able to degrade triflumezopyrim. CYPSF01 had an obviously co-expression relationship with nuclear receptor ultraspiracle (USP), which were all significantly up-regulated when exposed to triflumezopyrim. Further, a USP-binding motif MA0534.1 was enriched from the upregulated peaks by Assay for Transposase Accessible Chromatin (ATAC-seq) analysis, which exited in the peaks located on the promoter of CYPSF01; the yeast one-hybrid experiments confirmed that USP could bind to the CYPSF01 promoter. And the USP interference significantly down-regulated CYPSF01 expression, and resulted in the significantly increasing sensitivity to triflumezopyrim, its mortality rate increased 28.37%. Therefore, the overexpression of USP could cause to the overexpression of CYPSF01, ultimately resulting in the resistance to triflumezopyrim in S. furcifera.

Characterizing cytochrome P450 enzymes involved in plant apocarotenoid metabolism by using an engineered yeast system.

Cytochrome P450 enzymes (CYPs) are involved in metabolic steps that provide structural diversity during the biosynthesis of carotenoids and their oxidative cleavage products called apocarotenoids. Recent studies on bioactive apocarotenoids in plants revealed the necessity of performing further research to uncover the function of novel CYP enzymes that might be involved in apocarotenoid metabolism. We describe a series of in-vitro methods to characterize plant CYPs that metabolize apocarotenoids, using a specific Saccharomyces cerevisiae strain, WAT11, engineered to express a CYP redox partner, Arabidopsis thaliana NADPH-P450 reductase 1 (ATR1). This chapter provides protocols for construction and transformation of plasmids that express CYPs in yeast, isolation of yeast microsomes, and in-vitro enzymatic assays to validate the final metabolic products using LC-MS.

Cytochrome b 5 Binds Tightly to Several Human Cytochrome P450 Enzymes.

Numerous studies have been reported in the past 50-plus years regarding the stimulatory role of cytochrome b 5 (b 5) in some, but not all, microsomal cytochrome P450 (P450) reactions with drugs and steroids. A missing element in most of these studies has been a sensitive and accurate measure of binding affinities of b 5 with P450s. In the course of work with P450 17A1, we developed a fluorescent derivative of a human b 5 site-directed mutant, Alexa 488-T70C-b 5, that could be used in binding assays at sub-μM concentrations. Alexa 488-T70C-b 5 bound to human P450s 1A2, 2B6, 2C8, 2C9, 2E1, 2S1, 4A11, 3A4, and 17A1, with estimated K d values ranging from 2.5 to 61 nM. Only weak binding was detected with P450 2D6, and no fluorescence attenuation was observed with P450 2A6. All of the P450s that bound b 5 have some reported activity stimulation except for P450 2S1. The affinity of P450 3A4 for b 5 was decreased somewhat by the presence of a substrate or inhibitor. The fluorescence of a P450 3A4•Alexa 488-T70C-b 5 complex was partially restored by titration with NADPH-P450 reductase (POR) (K d,apparent 89 nM), suggesting the existence of a ternary P450 3A4-b 5-POR complex, as observed previously with P450 17A1. Gel filtration evidence was also obtained for this ternary complex with P450 3A4. Overall, the results indicated that the affinity of b 5 for many P450s is very high, and that ternary P450-b 5-POR complexes are relevant in P450 3A4 reactions as opposed to a shuttle mechanism. SIGNIFICANCE STATEMENT: High-affinity binding of cytochrome b 5 (b 5) (K d < 100 nM) was observed with many drug-metabolizing cytochrome P450 (P450) enzymes. There is some correlation of binding with reported stimulation, with several exceptions. Evidence is provided for a ternary P450 3A4-b 5-NADPH-P450 reductase complex.

Molecular bases of P450-mediated resistance to the neonicotinoid insecticide imidacloprid in the mosquito Ae. aegypti.

Resistance to chemical insecticides including pyrethroids, the main insecticide class used against mosquitoes, has re-kindled interest in the use of neonicotinoids. In this context, the present study aimed to characterize the molecular basis of neonicotinoid resistance in the mosquito Aedes aegypti. Resistance mechanisms were studied by combining transcriptomic and genomic data obtained from a laboratory strain selected at the larval stage after 30 generations of exposure to imidacloprid (Imida-R line). After thirty generations of selection, larvae of the Imida-R line showed an 8-fold increased resistance to imidacloprid and a significant cross-tolerance to the pyrethroids permethrin and deltamethrin. Cross-resistance to pyrethroids was only observed in adults when larvae were previously exposed to imidacloprid suggesting a low but inducible expression of resistance alleles at the adult stage. Resistance of the Imida-R line was associated with a slower larval development time in females. Multiple detoxification enzymes were over-transcribed in larvae in association with resistance including the P450s CYP6BB2, CYP9M9 and CYP6M11 previously associated with pyrethroid resistance. Some of them together with their redox partner NADPH P450 reductase were also affected by non-synonymous mutations associated with resistance. Combining genomic and transcriptomic data allowed identifying promoter variations associated with the up-regulation of CYP6BB2 in the resistant line. Overall, these data confirm the key role of P450s in neonicotinoid resistance in Ae. aegypti and their potential to confer cross-resistance to pyrethroids, raising concerns about the use of neonicotinoids for resistance management in this mosquito species.

Tight binding of cytochrome b5 to cytochrome P450 17A1 is a critical feature of stimulation of C21 steroid lyase activity and androgen synthesis

It has been recognized for >50 years that cytochrome b5 (b5) stimulates some cytochrome P450 (P450)–catalyzed oxidations, but the basis of this function is still not understood well. The strongest stimulation of catalytic activity by b5 is in the P450 17A1 lyase reaction, an essential step in androgen synthesis from 21-carbon (C21) steroids, making this an excellent model system to interrogate b5 function. One of the issues in studying b5–P450 interactions has been the limited solution assay methods. We constructed a fluorescently labeled variant of human b5 that can be used in titrations. The labeled b5 bound to WT P450 17A1 with a Kd of 2.5 nM and rapid kinetics, on the order of 1 s−1. Only weak binding was observed with the clinical P450 17A1 variants E305G, R347H, and R358Q; these mutants are deficient in lyase activity, which has been hypothesized to be due to attenuated b5 binding. Kd values were not affected by the presence of P450 17A1 substrates. A peptide containing the P450 17A1 Arg-347/Arg-358 region attenuated Alexa 488-T70C-b5 fluorescence at higher concentrations. The addition of NADPH–P450 reductase (POR) to an Alexa 488-T70C-b5:P450 17A1 complex resulted in a concentration-dependent partial restoration of b5 fluorescence, indicative of a ternary P450:b5:POR complex, which was also supported by gel filtration experiments. Overall, these results are interpreted in the context of a dynamic and tight P450 17A1:b5 complex that also binds POR to form a catalytically competent ternary complex, and variants that disrupt this interaction have low catalytic activity.

Functional interactions of adrenodoxin with several human mitochondrial cytochrome P450 enzymes

Seven of the 57 human cytochrome P450 (P450) enzymes are mitochondrial and carry out important reactions with steroids and vitamins A and D. These seven P450s utilize an electron transport chain that includes NADPH, NADPH-adrenodoxin reductase (AdR), and adrenodoxin (Adx) instead of the diflavin NADPH-P450 reductase (POR) used by the other P450s in the endoplasmic reticulum. Although numerous studies have been published involving mitochondrial P450 systems, the experimental conditions vary considerably. We compared human Adx and bovine Adx, a commonly used component, and found very similar catalytic activities in reactions catalyzed by human P450s 11B2, 27A1, and 27C1. Binding constants of 6–200 nM were estimated for Adx binding to these P450s using microscale thermophoresis. All P450 catalytic reactions were saturated at 10 μM Adx, and higher concentrations were not inhibitory up to at least 50 μM. Collectively these studies demonstrate the tight binding of Adx (both human and bovine) to AdR and to several mitochondrial P450s and provide guidance for optimization of Adx-dependent P450 reactions.

An Inexpensive, Efficient Alternative to NADPH to Support Catalysis by Thermostable Cytochrome P450 Enzymes

AbstractCytochrome P450 enzymes (P450s) are highly desirable catalysts for the regio‐ and stereo‐selective, late‐stage functionalization of pharmaceuticals and other fine chemicals. Recently, the resurrected ancestors of drug‐metabolizing P450s were shown to be highly thermostable and expressed in high yield, while retaining similar substrate specificity to the extant forms. However, they still rely on NADPH and cytochrome P450 reductase (CPR) to enable catalysis of oxidative transformations. To identify an alternative support system, we screened 10 oxygen surrogates (OSs) for the ability to support P450 ancestors from three different families. Of the 23 ancestors examined, 17 were supported by at least one OS as well as, or better than, by CPR. Using two candidate P450s we showed that OS‐dependent P450 catalysis can be optimized in a few steps, boosting product yield from ∼2.2 % with CPR to 88–100 % with an OS. The principles applied here will facilitate faster evaluation and optimization of OS‐supported P450 catalysis versus redox partner‐dependent P450 catalysis.

Effect of Sulfadimethoxine, Oxytetracycline, and Streptomycin Antibiotics in Three Types of Crop Plants—Root, Leafy, and Fruit

(1) Background: Plants act as the natural sink for a variety of toxins in the environment, including veterinary antibiotics (VAs). The objective of this study was to evaluate the uptake and fate of sulfadimethoxine (SDZ), oxytetracycline (OTC), and streptomycin (STR) in lettuce (Lactuca sativa L.), carrot (Daucus carota), and pepper (Capsicum annum) grown in VAs amended soil. (2) Methods: 0, 50, and 100 mg kg−1 VA laced manure was applied in a sandy clay loam soil. (3) Results: 30-d (lettuce) and 60-d (carrot and pepper) greenhouse experiment showed that SDZ and OTC were taken up by all three plants, with concentrations in plant tissue ranging from 0.1 to 1.2 mg kg−1 dry weight. The concentration of VAs in plant tissues increased with a corresponding increase of antibiotics in manure. The highest plant tissue concentrations were found in carrot and lettuce, followed by pepper. An increase in NADPH P450 reductase and glutathione-s-transferase enzyme activity with increasing SDZ and OTC concentration was evident, signifying the induction of the detoxification process. The activity of plant detoxification enzymes under STR treatment was found not to be significantly different from control. (4) Conclusions: These results raise potential human health concerns of consuming low levels of antibiotics from produce grown on manure-amended soils. The result indicates that SDZ, OTC, and STR antibiotics posed high, medium, and low acute ecological risks in lettuce, carrot, and pepper plants when grown in sandy clay loam soil.

Metabolic engineering of Yarrowia lipolytica for heterologous oleanolic acid production

Oleanolic acid (OA) is a pentacyclic triterpenoid known for its biological properties such as antioxidation activity. Herein, we developed an efficient biosynthetic pathway for OA production in Yarrowia lipolytica. The engineered strain, YL-OA0, produced 16.3 mg/L of OA, and the production efficiency was further increased by 5.65-fold to 92.1 mg/L following overexpression of the key genes involved in the mevalonate (MVA) pathway. Transcriptomic and metabolomic analyses demonstrated the increase in the percentage of unsaturated fatty acids. The electron transfer of the strain YL-OA7 was restricted. Therefore, we fused the gene encoding cytochrome P450 (CYP716A12) to that encoding NADPH-P450 reductase; this resulted in an increase in the OA yield to 129.9 mg/L in the strain YL-fuOA1. The strain YL-fuOA1 could produce 540.7 mg/L OA in fed-batch fermentation in a 5 L fermenter. This study shows the feasibility of the production of high value triterpenoid compounds using Y. lipolytica as a platform.

Inhibition of cytochrome P450 2B6 by Astragalus extract mixture HT042.

Astragalus extract mixture (AEM) HT042 is a functional food approved by the MFDS (Korean FDA) for increasing height. It comprises a mixture of three standardized extracts from Astragalus membranaceus root, Eleutherococcus senticosus stem, and Phlomis umbrosa root. In this study, drug-functional food interaction was analyzed using six major human cytochrome P450 enzymes. The inhibitory effect of AEM HT042 on P450 activities was studied using a P450-NADPH P450 reductase reconstitution system. Among the six P450 enzymes (1A2, 2A6, 2B6, 2D6, 2C9, and 3A4), only P450 2B6 activity was markedly decreased by AEM HT042 addition. The bupropion hydroxylation activity of P450 2B6 was analyzed using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). A calculated IC50 value of 10.62µg/ml was obtained. To identify the inhibitory compounds in the mixture, four active compounds in AEM HT042 were analyzed. Shanzhiside methylester exhibited inhibitory effects on P450 2B6, whereas formononetin, eleutheroside E, and sesamoside did not affect P450 2B6 activity at all. Our results suggest that shanzhiside methylester in AEM HT042 is responsible for the inhibitory effect on P450 2B6 metabolism. Characterization of the inhibitory effect on P450 can help determine the safe administration of functional foods along with many clinical drugs that aremetabolized by P450.

Viral-mediated gene delivery of TMBIM6 protects the neonatal brain via disruption of NPR-CYP complex coupled with upregulation of Nrf-2 post-HI

BackgroundOxidative stress, inflammation, and endoplasmic reticulum (ER) stress play a major role in the pathogenesis of neonatal hypoxic-ischemic (HI) injury. ER stress results in the accumulation of unfolded proteins that trigger the NADPH-P450 reductase (NPR) and the microsomal monooxygenase system which is composed of cytochrome P450 members (CYP) generating reactive oxygen species (ROS) as well as the release of inflammatory cytokines.We explored the role of Bax Inhibitor-1 (BI-1) protein, encoded by the Transmembrane Bax inhibitor Motif Containing 6 (TMBIM6) gene, in protection from ER stress after HI brain injury. BI-1 may attenuate ER stress-induced ROS production and release of inflammatory mediators via (1) disruption of the NPR-CYP complex and (2) upregulation of Nrf-2, a redox-sensitive transcription factor, thus promoting an increase in anti-oxidant enzymes to inhibit ROS production. The main objective of our study is to evaluate BI-1’s inhibitory effects on ROS production and inflammation by overexpressing BI-1 in 10-day-old rat pups.MethodsTen-day-old (P10) unsexed Sprague-Dawley rat pups underwent right common carotid artery ligation, followed by 1.5 h of hypoxia. To overexpress BI-1, rat pups were intracerebroventricularly (icv) injected at 48 h pre-HI with the human adenoviral vector-TMBIM6 (Ad-TMBIM6). BI-1 and Nrf-2 silencing were achieved by icv injection at 48 h pre-HI using siRNA to elucidate the potential mechanism. Percent infarcted area, immunofluorescent staining, DHE staining, western blot, and long-term neurobehavior assessments were performed.ResultsOverexpression of BI-1 significantly reduced the percent infarcted area and improved long-term neurobehavioral outcomes. BI-1’s mediated protection was observed to be via inhibition of P4502E1, a major contributor to ROS generation and upregulation of pNrf-2 and HO-1, which correlated with a decrease in ROS and inflammatory markers. This effect was reversed when BI-1 or Nrf-2 were inhibited.ConclusionsOverexpression of BI-1 increased the production of antioxidant enzymes and attenuated inflammation by destabilizing the complex responsible for ROS production. BI-1’s multimodal role in inhibiting P4502E1, together with upregulating Nrf-2, makes it a promising therapeutic target.

Enzymatic activation of indolequinone-substituted 5-fluorodeoxyuridine prodrugs in hypoxic cells

Among the various enzymes, reductases that catalyze one-electron reduction are involved in the selective activation of functional compounds or materials in hypoxia, which is one of the well-known pathophysiological characteristics of solid tumors. Enzymatic one-electron reduction has been recognized as a useful reaction that can be applied in the design of tumor hypoxia-targeting drugs. In this report, we characterized the enzymatic reaction of 5-fluorodeoxyuridine (FdUrd) prodrug bearing an indolequinone unit (IQ-FdUrd), which is a substrate of reductases. IQ-FdUrd was activated to release FdUrd under hypoxic conditions after treatment with cytochrome NADPH P450 reductase. We also confirmed that IQ-FdUrd showed selective cytotoxicity in hypoxic tumor cells.

In Vitro Approach to Addressing Violative Drug Residues in Cattle

Violative residues of the veterinary drug flunixin (FNX) are sometimes found in cattle that were presumably slaughtered in accordance with the withdrawal period prescribed by federal guidelines. A possible cause for violative residues may be slow rates of FNX metabolism associated with sex, age or illness of animals. Experiments were designed to investigate the effects of sex and age on rates of flunixin metabolism using liver microsomal, S9, cytosol, and homogenate fractions from heifers (n=5) and steers (n=6) less than 30 months of age, and cows (n=8) older than 48 months of age. Validation of subcellular fractions was achieved through enzyme analyses: cytochrome P450 (P450), NADPH‐P450 reductase and glucose‐6‐phosphate dehydrogenase (G6PDH) activities under optimized conditions. Similarly, conditions for flunixin metabolism were optimized and fractional activity and metabolite formation was determined. Cows had lower (P &lt; 0.05) concentrations of P450, NADPH‐P450 reductase activity, and FNX metabolism than their younger counterparts (heifers or steers). Using 15 min incubations, 5‐hydroxy FNX was the major metabolite detected by LC MS‐MS. Variables including exact animal age, cattle breed, diet, health and physiological status of animals at slaughter could have influenced results but were not assessed because of limitations associated with tissue collection.Support or Funding InformationAll funding of work was provided by USDA‐ARS.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Production of Triterpene Ginsenoside Compound K in the Non-conventional Yeast Yarrowia lipolytica.

Compound K (CK) is a rare, tetracyclic, triterpenoid compound with important medical properties, such as antitumor and anti-inflammatory activities. Herein, an efficient biosynthetic pathway of CK was constructed in metabolically engineered Yarrowia lipolytica for the first time, and the engineered strain, YL-CK0, produced 5.1 mg/L CK. The production of CK was further increased by 5.96-fold to 30.4 mg/L with overexpression of key genes in the MVA pathway and fusion of cytochrome P450 monooxygenase (PPDS) and NADPH-P450 reductase. Finally, 161.8 mg/L CK production was achieved by fed-batch fermentation in a 5 L fermenter using the strain YL-MVA-CK. To the best of our knowledge, this is the first report on heterologous CK synthesis with the highest titer in Y. lipolytica. This study demonstrates the feasibility of producing high-value triterpenoid compounds using Y. lipolytica as a platform.

Electrons released from both flavins of NADPH-P450 reductase contribute to the reductive mobilization of iron from ferritin.

Ferritin, an iron storage protein, plays an important role in iron homeostasis. The mechanism of reductive mobilization of iron from ferritin has not been clarified yet despite many studies. The aim of this study was to assess the mechanisms of the mobilization of iron from ferritin by NADPH P-450 reductase. Nucleotide-dependent flavoenzymes generated significant mobilization of iron from ferritin. The possibility of reductive mobilization of iron from ferritin by electrons released from flavin sites or heme site of two flavoenzymes was investigated to elucidate the mediator-independent mechanisms of such reductive mobilization. The mobilization by NADPH-P450 reductase in the presence of ferricyanide increased threefold, while in the presence of cytochrome C increased thirteen-fold. These results indicate that electrons released from both flavins of NADPH-P450 reductase contribute to the reductive mobilization of iron from ferritin. The mechanism of the mobilization of iron from ferritin is discussed. J. Med. Invest. 66 : 230-232, August, 2019.

In vitro subcellular characterization of flunixin liver metabolism in heifers, steers, and cows

The majority of cattle found to have violative liver residues of flunixin (FNX) in the United States are dairy cows. It has been hypothesized that illness of cows decreases the rate of FNX metabolism, resulting in violative residues at slaughter. Another contributing factor might be an age-related decrease in FNX metabolism, as dairy cull cows are typically older at slaughter than cattle raised for beef, rather than milk production. In order to investigate this possibility, subcellular fractions were prepared from liver slices from steers (n = 6) and heifers (n = 5) <30 months of age, and cows (n = 8) >48 mos of age. Cytochrome P450 (P450), NADPH-P450 reductase and glucose-6-phosphate dehydrogenase (G6PDH) activity and rate of 5-hydroxy FNX (5-OH FNX) formation were measured in liver homogenate, cytosolic, microsomal, and S9 fractions. Cows had lower concentrations of P450, NADPH-P450 reductase activity, and 5-OH FNX formation (P ≤ 0. 02), supporting the theory that advanced age may contribute to the higher incidence of violative FNX residues in dairy cows.