NADPH-cytochrome P450 Reductase Research Articles

Characterization of the structure and interactions of P450 BM3 using hybrid mass spectrometry approaches

The cytochrome P450 monooxygenase P450 BM3 (BM3) is a biotechnologically important and versatile enzyme capable of producing important compounds such as the medical drugs pravastatin and artemether, and the steroid hormone testosterone. BM3 is a natural fusion enzyme comprising two major domains: a cytochrome P450 (heme-binding) catalytic domain and a NADPH-cytochrome P450 reductase (CPR) domain containing FAD and FMN cofactors in distinct domains of the CPR. A crystal structure of full-length BM3 enzyme is not available in its monomeric or catalytically active dimeric state. In this study, we provide detailed insights into the protein-protein interactions that occur between domains in the BM3 enzyme and characterize molecular interactions within the BM3 dimer by using several hybrid mass spectrometry (MS) techniques, namely native ion mobility MS (IM-MS), collision-induced unfolding (CIU), and hydrogen-deuterium exchange MS (HDX-MS). These methods enable us to probe the structure, stoichiometry, and domain interactions in the ∼240 kDa BM3 dimeric complex. We obtained high-sequence coverage (88–99%) in the HDX-MS experiments for full-length BM3 and its component domains in both the ligand-free and ligand-bound states. We identified important protein interaction sites, in addition to sites corresponding to heme-CPR domain interactions at the dimeric interface. These findings bring us closer to understanding the structure and catalytic mechanism of P450 BM3.

Anti-stress phytohormones impact on proteome profile of green gram (Vigna radiata) under salt toxicity

Anti-stress phytohormones impact on proteome profile of green gram (Vigna radiata) under salt toxicity

Structure and Function of the Cytochrome P450 Monooxygenase Cinnamate 4-hydroxylase from Sorghum bicolor.

Cinnamate 4-hydroxylase (C4H; CYP73A) is a cytochrome P450 monooxygenase associated externally with the endoplasmic reticulum of plant cells. The enzyme uses NADPH-cytochrome P450 reductase as a donor of electrons and hydroxylates cinnamic acid to form 4-coumaric acid in phenylpropanoid metabolism. In order to better understand the structure and function of this unique class of plant P450 enzymes, we have characterized the enzyme C4H1 from lignifying tissues of sorghum (Sorghum bicolor), encoded by Sobic.002G126600 Here we report the 1.7 Å resolution crystal structure of CYP73A33. The obtained structural information, along with the results of the steady-state kinetic analysis and the absorption spectroscopy titration, displays a high degree of similarity of the structural and functional features of C4H to those of other P450 proteins. Our data also suggest the presence of a putative allosteric substrate-binding site in a hydrophobic pocket on the enzyme surface. In addition, comparing the newly resolved structure with those of well-investigated cytochromes P450 from mammals and bacteria enabled us to identify those residues of critical functional importance and revealed a unique sequence signature that is potentially responsible for substrate specificity and catalytic selectivity of C4H.

Protein profiling of chicken breast muscles from different slaughter houses in relation to meat quality

Introduction: Chicken meat is a source of protein in the human diet. Protein content and values define the quality of chicken meat. This research aimed to analyze variations of protein profiles in chicken breast muscles from different slaughtering houses by using proteomic strategies. Methods: Total proteins of chicken breast muscles from three different slaughtering houses (Sample A, Sample B and Sample C) were extracted and quantified by using Bradford assay. Then, the proteins were separated by SDS-PAGE to monitor the quality of extracted proteins. Protein profiles in different samples were compared by 2D-GE analysis. Results: The most highly expressed protein band was located between the molecular size of 37-50 kD in all samples and it was expected to be betaactin. While resolved in 2D-PAGE, differences in protein expression were observed between samples. There were three spots expressed with highest intensity in Sample B compared to others. The protein spot detected at pH 5.28 and the size range between 50- 75 kD was predicted to be NADPH-cytochrome P450 reductase (CPR), at pH 6.45 and molecular weight between 37-50 kD was expected to be creatine kinase M-type (M-CK) while at pH 6.78 and molecular size nearly 25 kD was expected to be αB-crystallin. Conclusions: It can be mentioned that these proteins could play a vital role in mechanisms that contribute to the poor quality of chicken meat.

Regions of CYP1A2 and CYP2B4 that are Involved in the Homomeric and Heteromeric Complex Formation: The Search Continues

Cytochromes P450 (CYP or P450) represent a superfamily of enzymes that interact with limited levels of NADPH‐cytochrome P450 reductase (POR) and are responsible for the oxidation of endogenous and exogenous compounds, converting them into water‐soluble products. This superfamily of heme‐containing enzymes are membrane‐bound and reside primarily in the endoplasmic reticulum (ER) of the liver, small intestine, and lung. As previously established, apart from interacting with limited levels of POR, the cytochrome P450s form homomeric and heteromeric complexes that alter the metabolic activities of the P450s involved. This study focuses on the interaction of CYP1A2 and CYP2B4. Previous work has shown that CYP1A2 is capable of forming homomeric and heteromeric complexes whereas CYP2B4 can form only heteromeric complexes. Simple binary systems containing CYP2B4 and POR exhibit typical Michaelis‐Menten saturation curves as a function of [POR], which is consistent with simple interactions between monomeric proteins. However, CYP1A2 activities for CYP1A2/POR binary systems exhibit a sigmoidal response as a function POR, which is consistent with cooperativity resulting from CYP1A2•CYP1A2 complexes. The heteromeric interaction of the two P450s causes the activity of CYP1A2 to increase and that of CYP2B4 to decrease. The objective of this study is to identify the surface regions of the proteins that interact in the CYP1A2•CYP1A2 and CYP1A2•CYP2B4 complexes. Using molecular cloning we substituted protein regions of CYP1A2 with regions of CYP2B4 and vice‐versa and expressed the chimera in the HEK‐293T cells as fusion proteins with C‐terminal protein tags that were capable of bioluminescence resonance energy transfer (BRET) when the chimeras interacted. We focused on the proximal face of the P450 as a potential region for P450•P450 complex formation. The proximal face is the region close to the heme that binds to the POR when electrons are transferred to the P450s. We have divided the proximal region into three regions designated as P1, P2, and P3. The amino acids L92‐K107 constituting P1, P132‐S156 representing P2 and T438‐K451 making up the P3 region of CYP1A2 were substituted with the respective regions of CYP2B4: T92‐S107 for P1, E132‐E163 for P2 and D438‐R448 for P3. The substitution of the P2 region of CYP1A2 with the corresponding region of CYP2B4 did not affect either CYP1A2•CYP1A2 or CYP1A2•CYP2B4 complex formation as indicated by the BRET measurements. Interestingly, the substitution of the P1 region of CYP1A2 with the corresponding region of CYP2B4 into the CYP1A2 protein disrupted the CYP1A2•CYP1A2 complex. This indicates that P1 is an important region involved in CYP1A2 homomeric complex formation. The role of P1 in the CYP1A2•CYP2B4 complex formation is currently being investigated along with the role of P3 in both the CYP1A2•CYP1A2 and the CYP1A2•CYP2B4 complexes.Support or Funding InformationSupported by NIH GM123253 & ES013648

Functional characterization of NADPH-cytochrome P450 reductase and cinnamic acid 4-hydroxylase encoding genes from Scoparia dulcis L.

BackgroundMost plant cytochrome P450 (P450) proteins need to be supplied with electrons from a redox partner, e.g. an NADPH-cytochrome P450 reductase (CPR), for the activation of oxygen molecules via heme. CPR is a flavoprotein with an N-terminal transmembrane domain, which transfers electrons from NADPH to the P450 via coenzymes flavin adenine dinucleotide and flavin mononucleotide.ResultsIn this study, a novel CPR (SdCPR) was isolated from a tropical medicinal plant Scoparia dulcis L. The deduced amino acid of SdCPR showed high homology of > 76% with CPR from higher plants and belonged to the class II CPRs of dicots. Recombinant SdCPR protein reduced cytochrome c, ferricyanide (K3Fe(CN)6), and dichlorophenolindophenol in an NADPH-dependent manner. To elucidate the P450 monooxygenase activity of SdCPR, we isolated a cinnamic acid 4-hydroxylase (SdC4H, CYP73A111) gene from S. dulcis. Biochemical characterization of SdCPR/SdC4H demonstrated that SdCPR supports the oxidation step of SdC4H. Real-time qPCR results showed that expression levels of SdCPR and SdC4H were inducible by mechanical wounding treatment and phytohormone elicitation (methyl jasmonate, salicylic acid), which were consistent with the results of promotor analyses.ConclusionsOur results showed that the SdCPR and SdC4H are related to defense reactions, including the biosynthesis of secondary metabolites.

RNA interference of NADPH-cytochrome P450 reductase increases susceptibilities to multiple acaricides in Tetranychus urticae

The two-spotted spider mite, Tetranychus urticae, is a polyphagous pest feeding on over 1100 plant species, including numerous highly valued economic crops. The control of T. urticae largely depends on the use of acaricides, which leads to pervasive development of acaricide resistance. Cytochrome P450-mediated metabolic detoxification is one of the major mechanisms of acaricide resistance in T. urticae. NADPH-cytochrome P450 reductase (CPR) plays as a crucial co-factor protein that donates electron(s) to microsomal cytochrome P450s to complete their catalytic cycle. This study seeks to understand the involvement of CPR/P450 in acaricide resistance in T. urticae. The full-length cDNA sequence of T. urticae's CPR (TuCPR) was cloned and characterized. TuCPR was ubiquitously transcribed in different life stages of T. urticae and the highest transcription was observed in the nymph and adult stages. TuCPR was constitutively over-expressed in six acaricide resistant populations compared to a susceptible one. TuCPR transcriptional expression was also induced by multiple acaricides in a time-dependent manner. Down-regulation of TuCPR via RNA interference (RNAi) in T. urticae led to reduced enzymatic activities of TuCPR and cytochrome P450s, as well as a reduction of resistance to multiple acaricides, abamectin, bifenthrin, and fenpyroximate. The outcome of this study highlights CPR as a potential novel target for eco-friendly control of T. urticae and other related plant-feeding pests.

Tolerance and bio-accumulation of aflatoxin B1 in invertebrate Litopenaeus vannamei and vertebrate Oreochromis niloticus

Aflatoxin B1 (AFB1) is a mycotoxin that is commonly detected in aquatic feed in tropical and subtropical regions. The toxic effects in shrimp and fish tissues following exposure to chronic AFB1 exposure has been studied but there is no information on tolerance between different species. This study was designed to compare the tolerance of AFB1 between invertebrate Litopenaeus vannamei and vertebrate Oreochromis niloticus. The emphasis was on growth profile, biotransformation, histopathology and AFB1 accumulation following exposure to increasing doses of AFB1. 182 shrimps and 146 tilapia were used and divided into control and AFB1 exposure groups. Shrimp were exposed to 1.5-fold increasing doses of 1.2, 1.8, 2.7, 4, 6 mg/kg AFB1 and tilapia to 3.2, 4.8, 7.2, 10.8, 16.2 mg/kg of AFB1 for 20 d with a 4-day exposure to each AFB1 with the shrimp/fish sacrificed on the last day of exposed dose(s). At each time point the controls were also sacrificed. A significant decrease in survival rate and weight gain (WG) was observed in shrimp. Higher AFB1 doses caused a decline in tilapia WG. Dose responsive AFB1 accumulation was evident in muscle and shrimp hepatopancreas /tilapia liver. The concentration of AFB1 was significantly higher in shrimp hepatopancreas (22.76–72.89 ng/g) than in tilapia liver (6.68–19.45 ng/g) in spite of exposure to a higher dose regime. The muscle AFB1 concentration in both species ranged from 0 to 20 ng/g. The shrimp hepatopancreas cytochrome b5 (Cyt b5) concentration increased initially but declined after >4 mg/kg AFB1 exposure. The tilapia liver Cyt b5 content declined after >10.8 mg/kg AFB1. In the shrimp hepatopancreas, a marked induction of enzymes, aniline hydroxylase (AH), NADPH-cytochrome P450 reductase (NCCR), 7-ethoxyresorufin O-deethylase (EROD), glutathione-S-transferase (GST), sulfotransferase (SULT) and uridinediphosphate glucuronyltransferase (UGT) were observed. Only some of these enzymes, namely, EROD, GST, UGT and SULT but not AH or NCCR were induced in tilapia liver. Hepatopancreas/liver damage in shrimp/fish was marked at higher AFB1 doses. Marked changes in the shrimp hepatopancreatic cell structure was observed in shrimp at concentrations >6 mg/kg AFB1. This study showed that shrimp are more susceptible to AFB1 than tilapia and further studies are required to determine the role of AH and NCCR enzymes in species differences to AFB1 toxicity.

Synthetic Derivatives of (+)-epi-α-Bisabolol Are Formed by Mammalian Cytochromes P450 Expressed in a Yeast Reconstituted Pathway

Identification of the enzyme(s) involved in complex biosynthetic pathways can be challenging. An alternative approach might be to deliberately diverge from the original natural enzyme source and use promiscuous enzymes from other organisms. In this paper, we have tested the ability of a series of human and animal cytochromes P450 involved in xenobiotic detoxification to generate derivatives of (+)-epi-α-bisabolol and attempt to produce the direct precursor of hernandulcin, a sweetener from Lippia dulcis for which the last enzymatic steps are unknown. Screening steps were implemented in vivo in S. cerevisiae optimized for the biosynthesis of oxidized derivatives of (+)-epi-α-bisabolol by coexpressing two key enzymes: the (+)-epi-α-bisabolol synthase and the NADPH cytochrome P450 reductase. Five out of 25 cytochromes P450 were capable of producing new hydroxylated regioisomers of (+)-epi-α-bisabolol. Of the new oxidized bisabolol products, the structure of one compound, 14-hydroxy-(+)-epi-α-bisabolol, was fully elucidated by NMR while the probable structure of the second product was determined. In parallel, the production of (+)-epi-α-bisabolol derivatives was enhanced through the addition of a supplementary genomic copy of (+)-epi-α-bisabolol synthase that augmented the final titer of hydroxylated product to 64 mg/L. We thus demonstrate that promiscuous drug metabolism cytochromes P450 can be used to produce novel compounds from a terpene scaffold.

NADPH-cytochrome P450 reductase expression and enzymatic activity in primary-like human hepatocytes and HepG2 cells for in vitro biotransformation studies.

BACKGROUND:Human hepatocyte in vitro cell culture systems are important models for drug development and toxicology studies in the context of liver xenobiotic metabolism. Often, such culture systems are used to elucidate the biotransformation of xenobiotics or drugs and further investigate drug and drug metabolite effects on biological systems in terms of potential therapeutic benefit or toxicity. Human hepatocytes currently used for such in vitro studies are mostly primary cells or cell lines derived from liver cancers. Both approaches have limitations such as low proliferation capacity and progressive dedifferentiation found in primary cells or lack of liver functions in cell lines, which makes it difficult to reliably predict biotransformation of xenobiotics in patients. In order to overcome these limitations, HepaFH3 cells and Upcyte® hepatocytes representing primary-like hepatocytes of the first and second generation are increasingly used. Based on primary human hepatocyte cells transduced for stable expression of Upcyte® proliferation genes, they are mitotically active and exhibit liver functions over an extended period, making them comparable to primary human hepatocytes. These hepatocyte models show active liver metabolism such as urea and glycogen formation as well as biotransformation of xenobiotics. The latter is based on the expression, activity and inducibility of cytochrome P450 enzymes (CYP) as essential phase I reaction components. However, for further characterisation in terms of performance and existing limitations, additional studies are needed to elucidate the mechanisms involved in phase I reactions. One prerequisite is sufficient activity of microsomal NADPH-cytochrome P450 reductase (POR) functionally connected as electron donor to those CYP enzymes.OBJECTIVE:For Upcyte® hepatocytes and HepaFH3 cells, it is so far unknown to what extent POR is expressed, active, and may exert CYP-modulating effects. Here we studied POR expression and corresponding enzyme activity in human hepatoblastoma cell line HepG2 and compared this with HepaFH3 and Upcyte® hepatocytes representing proliferating primary-like hepatocytes.METHODS:POR expression of those hepatocyte models was determined at mRNA and protein level using qRT-PCR, Western Blot and immunofluorescence staining. Kinetic studies on POR activity in isolated microsomes were performed by a colorimetric method.RESULTS:The investigated hepatocyte models showed remarkable differences at the level of POR expression. Compared to primary-like hepatocytes, POR expression of HepG2 cells was 4-fold higher at mRNA and 2-fold higher at protein level. However, this higher expression did not correlate with corresponding enzyme activity levels in isolated microsomes, which were comparable between all cell systems tested. A tendency of higher POR activity in HepG2 cells compared to HepaFH3(p = 0.0829) might be present. Compared to primary human hepatocyte microsomes, POR activity was considerably lower in all hepatocyte models.CONCLUSION:In summary, our study revealed that POR expression and activity were clearly detectable in all in vitro hepatocyte models with the highest POR expression in cancer cell line HepG2. However, POR activity was lower in tested hepatocyte models when compared to human primary hepatocyte microsomes. Whether this was caused by e.g. polymorphisms or metabolic differences of investigated hepatocyte models will be target for future studies.

Proximal Tubular Vacuolization and Hypersensitivity to Drug-Induced Nephrotoxicity in Male Mice With Decreased Expression of the NADPH-Cytochrome P450 Reductase.

The effect of variations in the expression of cytochrome P450 reductase (CPR or POR) is determined in mice with decreased POR expression to identify potential vulnerabilities in people with low POR expression. There is an age-dependent appearance of increasing vacuolization in the proximal tubules of the renal cortex in 4- to 9-month-old male (but not female) Cpr-low (CL) mice. These mice have low POR expression in all cells of the body and upregulation of lysosome-associated membrane protein 1 expression in the renal cortex. Vacuolization is also seen in extrahepatic CL and extrarenal CL male mice, but not in mice with tissue-specific Por deletion in liver, intestinal epithelium, or kidney. The occurrence of vacuolization is accompanied by increases in serum blood-urea-nitrogen levels. Male CL mice are hypersensitive to cisplatin- and gentamicin-induced renal toxicity at 3 months of age, before proximal tubular (PT) vacuoles are detectable. At doses that do not cause renal toxicity in wild-type mice, both drugs cause substantial increases in serum blood-urea-nitrogen levels and PT vacuolization in male but not female CL mice. The hypersensitivity to drug-induced renal toxicity is accompanied by increases in circulating drug levels. These novel findings demonstrate deficiency of renal function in mice with globally reduced POR expression and suggest that low POR expression may be a risk factor for drug-induced nephrotoxicity in humans.

Molecular characterization of an NADPH cytochrome P450 reductase from Bemisia tabaci Q: Potential involvement in susceptibility to imidacloprid

NADPH cytochrome P450 reductase (CPR) is an integral component of cytochrome P450-mediated biological reactions, such as the metabolism of xenobiotics, including insecticides. CPR has been reported to be associated with insecticide tolerance in several insects. However, the biochemical characteristics and biological function of CPR in Bemisia tabaci Q (BtCPR) remain undefined. In this study, BtCPR was cloned, and bioinformatic analysis showed that BtCPR is a transmembrane protein with a molecular weight (MW) of 76.73 kDa and contains conserved binding domains (FMN, FAD, and NADPH). Tissue- and developmental stage-dependent expression indicated that the highest expression levels of BtCPR occurred in head tissue and in male adults. Transcripts of BtCPR in the field B. tabaci Q strain were 1.62-fold higher than those of the laboratory B. tabaci Q strain. In both field and laboratory adults, the susceptibility of BtCPR-knockdown B. tabaci Q to imidacloprid substantially increased compared to that of the B. tabaci Q control group. Furthermore, the heterologous expression of BtCPR in Sf9 cells exhibited catalytic activity for cytochrome c reduction, following Michaelis-Menten kinetics. Sf9 cells overexpressing BtCPR had greater viability than the control cells when treated with imidacloprid. The results suggest that BtCPR could affect the susceptibility of B. tabaci Q to imidacloprid and could also be considered a novel target for pest control.

Knockdown of NADPH-cytochrome P450 reductase and CYP6MS1 increases the susceptibility of Sitophilus zeamais to terpinen-4-ol

Terpinen-4-ol showed highly insecticidal activity to stored-grain pest Sitophilus zeamais, and cytochrome P450s were strongly induced in response to terpinen-4-ol fumigation. Understanding of the function of P450 enzyme system in the susceptibility to terpinen-4-ol in S. zeamais will benefit the potential application of terpinen-4-ol in controlling stored-grain pests. In the present study, the synergist piperonyl butoxide increased the toxicity of terpinen-4-ol to S. zeamais, with a synergism ratio of 3.5-fold. Two isoforms of NADPH-cytochrome P450 reductase (SzCPR) were identified, with the difference at the N-terminal. SzCPR contained an N-terminal membrane anchor, FMN, FAD, and NADP binding domains. Expression levels of SzCPR were upregulated by tea tree oil (TTO) and its main constituent terpinen-4-ol under different concentrations and time periods. RNAi was generated for S. zeamais by feeding adults dsRNA and the knockdown of SzCPR increased the susceptibility of S. zeamais to terpinen-4-ol, with higher mortality of adults than control under terpinen-4-ol fumigation. Further RNAi analysis showed that P450 gene CYP6MS1 mediated the susceptibility of S. zeamais to terpinen-4-ol. These results revealed that cytochrome P450 enzyme system, especially CYP6MS1 participated in the susceptibility of S. zeamais to terpinen-4-ol. The findings provided a foundation to clarify the metabolic mechanisms of terpinen-4-ol in stored-grain pests.

Short-lived neutral FMN and FAD semiquinones are transient intermediates in cryo-reduced yeast NADPH-cytochrome P450 reductase

The electron configuration of flavin cofactors, FMN and FAD, is a critical factor governing the reactivity of NADPH-cytochrome P450 reductase (CPR). The current view of electron transfer by the mammalian CPR, based on equilibrium redox potentials of the flavin cofactors, is that the two electron-reduced FMN hydroquinone (FMNH2), rather than one electron-reduced FMN semiquinone, serves as electron donor to the terminal protein acceptors. However, kinetic and thermodynamic studies on the CPR species originated from different organisms have shown that redox potentials measured at distinct electron transfer steps differ from redox potentials determined by equilibrium titration. Collectively, previous observations suggest that the short-lived transient semiquinone species may carry electrons in diflavin reductases. In this work, we have investigated spectroscopic properties of the CPR-bound FAD and FMN reduced at 77 K by radiolytically-generated thermalized electrons. Using UV–vis spectroscopy, we demonstrated that upon cryo-reduction of oxidized yeast CPR (yCPR) containing an equimolar ratio of both FAD and FMN, or FAD alone, neutral semiquinones were trapped at 77 K. During annealing at the elevated temperatures, unstable short-lived neutral semiquinones relaxed to spectroscopically distinct air-stable neutral semiquinones. This transition was independent of pH within the 6.0–10.7 range. Our data on yeast CPR are in line with the previous observations of others that the flavin short-lived transient semiquinone intermediates may have a role in the electron transfer by CPR at physiological conditions.

Tobacco Cutworm (Spodoptera Litura) Larvae Silenced in the NADPH-Cytochrome P450 Reductase Gene Show Increased Susceptibility to Phoxim.

Nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductases (CPRs) function as redox partners of cytochrome P450 monooxygenases (P450s). CPRs and P450s in insects have been found to participate in insecticide resistance. However, the CPR of the moth Spodoptera litura has not been well characterized yet. Based on previously obtained transcriptome information, a full-length CPR cDNA of S. litura (SlCPR) was PCR-cloned. The deduced amino acid sequence contains domains and residues predicted to be essential for CPR function. Phylogenetic analysis with insect CPR amino acid sequences showed that SlCPR is closely related to CPRs of Lepidoptera. Quantitative reverse transcriptase PCR (RT-qPCR) was used to determine expression levels of SlCPR in different developmental stages and tissues of S. litura. SlCPR expression was strongest at the sixth-instar larvae stage and fifth-instar larvae showed highest expression in the midgut. Expression of SlCPR in the midgut and fat body was strongly upregulated when fifth-instar larvae were exposed to phoxim at LC15 (4 μg/mL) and LC50 (20 μg/mL) doses. RNA interference (RNAi) mediated silencing of SlCPR increased larval mortality by 34.6% (LC15 dose) and 53.5% (LC50 dose). Our results provide key information on the SlCPR gene and indicate that SlCPR expression levels in S. litura larvae influence their susceptibility to phoxim and possibly other insecticides.

Rational evolution of the cofactor-binding site of cytochrome P450 reductase yields variants with increased activity towards specific cytochrome P450 enzymes.

NADPH-cytochrome P450 reductase (CPR) is the natural redox partner of microsomal cytochrome P450 enzymes. CPR shows a stringent preference for NADPH over the less expensive cofactor, NADH, economically limiting its use as a biocatalyst. The complexity of cofactor-linked CPR protein dynamics and the incomplete understanding of the interaction of CPR with both cofactors and electron acceptors present challenges for the successful rational engineering of a CPR with enhanced activity with NADH. Here, we report a rational evolution approach to enhance the activity of CPR with NADH, in which mutations were introduced into the NADPH-binding flavin adenine dinucleotide (FAD) domain. Multiple CPR mutants that used NADH more effectively than the wild-type CPR in the reduction of the surrogate electron acceptor, cytochrome c were found. However, most were inactive in supporting P450 activity, arguing against the use of cytochrome c as a surrogate electron acceptor. Unexpectedly, several mutants showed significantly improved activity towards CYP2C9 (mutant 1-014) and/or CYP2A6 (mutants 1-014, 1-015, 1-053 and 1-077) using NADPH, even though the mutations were introduced at locations remote from the putative CPR-P450 interaction face. Therefore, mutations at sites in the FAD domain of CPR may be promising future engineering targets to enhance P450-mediated substrate turnover. ENZYMES: NADPH-cytochrome P450 reductase - EC 1.6.2.4; cytochrome P450 - EC 1.14.14.1.

Optimization of the process parameters for reduction of gossypol levels in cottonseed meal by functional recombinant NADPH-cytochrome P450 reductase and cytochrome P450 CYP9A12 of Helicoverpa armigera

Gossypol is a toxic polyphenolic product that is derived from cotton plants. The toxicity of gossypol has limited the utilization of cottonseed meal (CSM) in the feed industry. The gene, Helicoverpa armigera CYP9A12, is a gossypol-inducible cytochrome P450 gene. The objective of our study was to obtain the functional recombinant H. armigera CYP9A12 enzyme in Pichia pastoris and to verify whether this candidate enzyme could decrease gossypol in vitro. Free and total gossypol contents were detected in the enzyme solution and in CSM. The H. armigera CYP9A12 enzyme degraded free concentration of gossypol. After optimization of the single-test and response surface method, free gossypol content could be decreased to 40.91 mg/kg in CSM by the H. armigera CYP9A12 enzyme when the initial temperature was 35 °C, the enzymatic hydrolysis time lasted 2.5 h, the enzyme addition was 2.5 mL, and the substrate moisture was 39%.

Efficient production of glycyrrhetinic acid in metabolically engineered Saccharomyces cerevisiae via an integrated strategy

BackgroundGlycyrrhetinic acid (GA) is the most important ingredient in licorice due to its outstanding anti-inflammatory activity and wide application in the medicine and cosmetics industries. Contemporary industrial production of GA by acid hydrolysis of glycyrrhizin which was extracted from Glycyrrhiza plants, is not environment-friendly and devastates farmland since the Glycyrrhiza rhizomes grow up to 10 m underground.ResultsIn this study, GA was produced through metabolically engineering Saccharomyces cerevisiae by introducing the entire heterogeneous biosynthetic pathway of GA. Codon optimized CYP88D6 and CYP72A154, combined with β-AS (β-amyrin synthase encoding gene) and the NADPH-cytochrome P450 reductase gene of Arabidopsis thaliana were introduced into S. cerevisiae. The resulting strain (Y1) produced 2.5 mg/L of β-amyrin and 14 μg/L of GA. The cytochrome b5 from G. uralensis (GuCYB5) was identified and the introduction of this novel GuCYB5 increased the efficiency of GA production by eightfold. The joint utilization of the GuCYB5 gene along with 10 known MVA pathway genes from S. cerevisiae were overexpressed in a stable chromosome integration to achieve higher GA production. Using the combined strategy, GA concentration improved by 40-fold during batch fermentation. The production was further improved to 8.78 mg/L in fed-batch fermentation, which was increased by a factor of nearly 630.ConclusionsThis study first investigated the influence of carbon flux in the upstream module and the introduction of a newly identified GuCYB5 on GA production. The newly identified GuCYB5 was highly effective in improving GA production. An integrated strategy including enzyme discovery, pathway optimization, and fusion protein construction was provided in improving GA production, achieving a 630 fold increase in GA production. The metabolically engineered yeast cell factories provide an alternative approach to glycyrrhetinic acid production, replacing the traditional method of plant extraction.

Boosting the biosynthesis of betulinic acid and related triterpenoids in Yarrowia lipolytica via multimodular metabolic engineering

BackgroundBetulinic acid is a pentacyclic lupane-type triterpenoid and a potential antiviral and antitumor drug, but the amount of betulinic acid in plants is low and cannot meet the demand for this compound. Yarrowia lipolytica, as an oleaginous yeast, is a promising microbial cell factory for the production of highly hydrophobic compounds due to the ability of this organism to accumulate large amounts of lipids that can store hydrophobic products and supply sufficient precursors for terpene synthesis. However, engineering for the heterologous production of betulinic acid and related triterpenoids has not developed as systematically as that for the production of other terpenoids, thus the production of betulinic acid in microbes remains unsatisfactory.ResultsIn this study, we applied a multimodular strategy to systematically improve the biosynthesis of betulinic acid and related triterpenoids in Y. lipolytica by engineering four functional modules, namely, the heterogenous CYP/CPR, MVA, acetyl-CoA generation, and redox cofactor supply modules. First, by screening 25 combinations of cytochrome P450 monooxygenases (CYPs) and NADPH-cytochrome P450 reductases (CPRs), each of which originated from 5 different sources, we selected two optimal betulinic acid-producing strains. Then, ERG1, ERG9, and HMG1 in the MVA module were overexpressed in the two strains, which dramatically increased betulinic acid production and resulted in a strain (YLJCC56) that exhibited the highest betulinic acid yield of 51.87 ± 2.77 mg/L. Then, we engineered the redox cofactor supply module by introducing NADPH- or NADH-generating enzymes and the acetyl-CoA generation module by directly overexpressing acetyl-CoA synthases or reinforcing the β-oxidation pathway, which further increased the total triterpenoid yield (the sum of the betulin, betulinic acid, betulinic aldehyde yields). Finally, we engineered these modules in combination, and the total triterpenoid yield reached 204.89 ± 11.56 mg/L (composed of 65.44% betulin, 23.71% betulinic acid and 10.85% betulinic aldehyde) in shake flask cultures.ConclusionsHere, we systematically engineered Y. lipolytica and achieved, to the best of our knowledge, the highest betulinic acid and total triterpenoid yields reported in microbes. Our study provides a suitable reference for studies on heterologous exploitation of P450 enzymes and manipulation of triterpenoid production in Y. lipolytica.

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.