ABSTRACT
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.
Subject(s)
NADPH-Ferrihemoprotein Reductase , Cloning, Organism , Brachyura , Gene Expression , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.