Cloning, expression and functional identification of cytochrome P450 reductase gene in Aquilaria sinensis / 药学学报

Cytochrome P450 reductase (CPR) is essential for the electron transport chain of cytochrome P450s, playing an indispensable role in electron transfer in vivo. In this study, one cDNA encoding cytochrome P450 reductase (Ascpr1) was identified from the callus of Aquilaria sinensis. Ascpr1 contains an open reading frame of 2 124 bp. The deduced protein is composed of 707 amino acids, with a predicted molecular weight of 78.82 kD. Phylogenetic analysis revealed that AsCPR1 is a type Ⅱ CPR protein closely related to the CPR from Theobroma cacao. Transmembrane prediction using TMHMM 2.0 indicated that the amino acids 52-71 of AsCPR1 comprise a transmembrane region. After truncating of 67 amino acid residues from N-terminal, the truncated AsCPR1 was successfully expressed in E. coli Transetta (DE3). Further purification of the recombinant AsCPR1 by affinity chromatography and determination of the enzymatic activity allowed the reducing ability of AsCPR1 to cytochrome C in vitro. The results pave the way for further study on the synthesis of defensive chemicals involved in P450s and the functions of CPR in self-defense of A. sinensis.

Cloning and Expression of NADPH-cytochrome P450 Reductase Gene in Chinese Mitten Crab, Eriocheir sinensis

Abstract NADPH-cytochromeP450 reductase (CPR) is one of the most important components of the cytochrome P450 enzyme system. In this study, a gene encoding CPR (named EsCPR) was isolated from Eriocheir sinensis using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. Analysis of the nucleotide sequence revealed a cDNA full-length of 3717 bp with an open reading frame of 2046 bp, a 5′-untranslated region of 42 bp, and a long 3′-untryganslated region of 1628bp, which encodes a protein of 681 amino acids with a predicted molecular weight of 30.7 kDa and an estimated pI of 4.82. The mature peptide shares amino acid of E. sinensis identity 82 % - 89 % to the CPR from Penaeus vannamei and Chionoecetes opilio. Tissues and developmental stage-dependent expression of EsCPR mRNA was investigated by real-time quantitative PCR. EsCPR mRNA was markedly expressed in the hepatopancreas and stomach. These results would provide valuable information for further study on the interactions between CPR and cytochrome P450 enzyme systems.

Ginsenosides in

Ginsenosides are a series of glycosylated triterpenoids which belong to protopanaxadiol (PPD)-, protopanaxatriol (PPT)-, ocotillol (OCT)- and oleanane (OA)-type saponins known as active compounds of

Cloning and related bioinformatics analysis of new cytochrome NADPH P450 reductase gene in Glycyrrhiza uralensis / 中草药

Objective To clone a new NADPH cytochrome P450 reductase gene (GuCPR) (Login number: MH401048) from Glycyrrhiza uralensis and do some bioinformatics analysis. Methods Total RNA was extracted from the roots of G. uralensis and then transcription reversed into cDNA. The GuCPR gene was obtained by screening the transcriptome database. The NCBI ORF finder was used to obtain its open reading frame and translated amino acid sequence, design primers for PCR amplification, construction of recombinant cloned plasmid. Bioinformatics analysis was used to predict the protein properties, structure and model the tertiary structure of the protein, and homologous phylogenetic tree construction was performed. Results cDNA of GuCPR gene was cloned to a total length of 2 118 bp and encoded 705 amino acid residues with a relative molecular mass of 78 450. Electricity (pI) 5.19. GuCPR protein was a non-secretory protein and did not have a signal recognition function. The results of transmembrane prediction showed that the amino acids 44—64 of the protein were transmembrane regions. Meanwhile, the subcellular localization prediction results showed that the protein was located on the endoplasmic reticulum. Conclusion A new GuCPR was cloned and its bioinformatics analysis laid the foundation for further research.

Cloning and functional identification of a new NADPH-cytochrome P450 reductase in Andrographis paniculata / 中国中药杂志

Andrographolide is a main active ingredient in traditional Chinese medicine Andrographis paniculata，with a variety of pharmacological activity，widely used in clinical practice. However its biosynthetic pathway has not been resolved. Cytochrome P450 reductase provides electrons for CYP450 and plays an important role in the CYP450 catalytic process. In this study，the coding sequence of A. paniculata CPR was screened and cloned by homologous alignment，named ApCPR4. The ApCPR4 protein was obtained by prokaryotic expression. After isolation and purification，the enzyme activity was identified . The results showed that ApCPR4 could reduce the cytochrome c and ferricyanide in NADPH-dependent manner. In order to verify its function，ApCPR4 and CYP76AH1 were co-transformed into yeast engineering bacteria. The results showed that ApCPR4 could help CYP76AH1 catalyze the formation of rustols in yeast. Real-time quantitative PCR results showed that the expression of ApCPR4 increased gradually in leaves treated with methyl jasmonate (MeJA). The expression pattern was consistent with the trend of induction and accumulation of andrographolide by MeJA，suggesting that ApCPR4 was associated with biosynthesis of andrographolide.

Expression and characterization of NADPH-cytochrome P450 reductase from Trametes versicolor in Escherichia coli / 生物工程学报

Trametes versicolor has strong ability to degrade environmental organic pollutants. NADPH-cytochrome P450 reductase (CPR) of T. versicolor transfers electron to cytochrome P450s (CYPs) and participates in the degradation process of organic pollutants. Sequence analysis showed that the genome of T. versicolor contains 1 potential CPR and multiple potential CYP sequences. To further study the molecular mechanism for the involvement of T. versicolor CPR in the cellular degradation of organic pollutants, a CPR gene from T. versicolor was cloned and heterologously expressed in Escherichia coli. Subsequently, the main properties of the recombinant enzyme were investigated. A truncated CPR protein lacking the predicted membrane anchor region (residues 1-24), named CPRΔ24, was overexpressed as a soluble form in E. coli. The recombinant CPRΔ24 protein showed a molecular weight consistent with the theoretical value of 78 kDa. Recombinant CPRΔ24 was purified using a Ni²⁺-chelating column followed by size exclusion chromatography. The specific activity of the purified CPRΔ24 was 5.82 U/mg. The CPRΔ24 enzyme displayed the maximum activity at 35 ℃ and pH 8.0. It has different degrees of tolerance against several types of metal ions and organic solvents. The apparent Km and kcat values of recombinant CPRΔ24 for NADPH were 19.7 μmol/L and 3.31/s, respectively, and those for the substrate cytochrome c were 25.9 μmol/L and 10.2/s, respectively, under conditions of 35 ℃ and pH 8.0. The above research provides the basis for exploring the functional mechanism of T. versicolor CPR in the degradation pathway of environmental organic pollutants.

Cloning and bioinformatics analysis of cytochrome P450 reductase gene in Poria cocos / 中草药

Objective: To clone cytochrome P450 reductase (PcCPR) gene from Poria cocos and to characterize with bioinformatics methods. Methods: According to annotated transcriptome of P. cocos, the PcCPR gene was cloned through RACE, and the genomic DNA sequence was further obtained through PCR. The characteristics of the encoded protein were analyzed using bioinformatics, the 3D structure of the protein was modeled with I-TASSER server, and phylogenetic tree of CPR was carried out with MEGA. Results: The 2 514 bp full-length cDNA sequence (GenBank Accession No. KP768251) and the 5 292 bp genomic DNA sequence (GenBank Accession No. KP896487) of PcCPR was obtained, which contained four exons and three introns. PcCPR encoded a protein with 732 amino acids. The protein was predicted to be an unstable hydrophilic protein with calculated molecular weight of 81 147 and isoelectric point 5.39. PcCPR does not have a signal peptide but has a transmembrane segment (aa residues 7 to 22), and it is anchored to endoplasmic reticulum. There are two flavin binding domains and many predicted FAD and NADP binding sites, these sites are adjacent respectively at 3D structure level. The homologous analysis indicates that PcCPR has a higher similarity with CPR from basidiomycetes than CPR from ascomycetes. Conclusion: The PcCPR was successfully cloned, which will provide a foundation for researches on PcCPR and PcCPR associated metabolic.

An update on the role of intestinal cytochrome P450 enzymes in drug disposition

Oral administration is the most commonly used route for drug treatment. Intestinal cytochrome P450 (CYP)-mediated metabolism can eliminate a large proportion of some orally administered drugs before they reach systemic circulation, while leaving the passage of other drugs unimpeded. A better understanding of the ability of intestinal P450 enzymes to metabolize various clinical drugs in both humans and preclinical animal species, including the identification of the CYP enzymes expressed, their regulation, and the relative importance of intestinal metabolism compared to hepatic metabolism, is important for improving bioavailability of current drugs and new drugs in development. Here, we briefly review the expression of drug-metabolizing P450 enzymes in the small intestine of humans and several preclinical animal species, and provide an update of the various factors or events that regulate intestinal P450 expression, including a cross talk between the liver and the intestine. We further compare various clinical and preclinical approaches for assessing the impact of intestinal drug metabolism on bioavailability, and discuss the utility of the intestinal epithelium-specific NADPH-cytochrome P450 reductase-null (IECN) mouse as a useful model for studyingroles of intestinal P450 in the disposition of orally administered drugs.

Research progresses of cytochrome P450 reductase / 中国药理学通报

Cytochrome P450 reductase(CPR) is a kind of membrane protein which transfer electrons to cytochrome P450s in the system of drug metabolism.The structure,substrate-fuction and gene modulating of CPR is concluded,and the questions of functional mechanism of CPR are discussed in this article.