Part:BBa_K5327010
NADPH--cytochrome P450 reductase 2
This enzyme is required for electron transfer from NADP to cytochrome P450 in microsomes. It can also provide electron transfer to heme oxygenase and cytochrome B5. Reduces a variety of substrates in vitro, such as cytochrome c, feericyanide and dichloroindophenol.
Usage and Biology
Genome localization:Chromosome 4 - NC_003075.7
Expression diagram:
- Fig 1. The expression diagram of NADPH--cytochrome P450 reductase 2
Corresponding enzyme structure:
- Fig 2. The corresponding enzyme structure of NADPH--cytochrome P450 reductase 2
The PCR result:
- Fig 3. The PCR result of NADPH--cytochrome P450 reductase 2
Subcellular localization:[4]
Located in the endoplasmic reticulum membrane of cells
- Fig 4. The subcellular localization of NADPH--cytochrome P450 reductase 2
Dynamics data:
- Table 1. The dynamics data of NADPH--cytochrome P450 reductase 2
Design Notes
The NADPH–cytochrome P450 reductase 2 (ATR2) gene was designed based on the coding sequence (CDS) from Arabidopsis thaliana and codon-optimized for expression in Saccharomyces cerevisiae (S288C) to ensure efficient expression. ATR2 encodes a flavoprotein that catalyzes the transfer of electrons from NADPH to various cytochrome P450 proteins, enabling these enzymes to activate dioxygen for oxidation reactions.[5]In this experiment, the enzyme provides electron transfer not only to cytochrome P450 but also interacts with cytochrome b5, assisting its function.[6]Additionally, ATR2 can reduce a range of substrates, including cytochrome c, ferricyanide, and dichlorophenol indophenol, demonstrating its broad substrate reduction capability. To achieve high expression levels and mRNA stability, the gene is expressed using the ADH1 promoter (ADH1pBBa_J63005) and TDH2 terminator (TDH2tBBa_K2407108). The optimized gene was inserted into a vector and introduced into Saccharomyces cerevisiae S288C via homologous recombination, followed by screening and expression verification in a deficient strain. This design aims to enhance the expression of ATR2 in yeast, thereby improving the activity of cytochrome P450 and optimizing yeast as a metabolic engineering platform.
Plasmid
- Fig 5. The plasmid expression of NADPH--cytochrome P450 reductase 2
Source
Arabidopsis thaliana
References
- ↑ HULL A K, CELENZA J L. Bacterial expression and purification of the Arabidopsis NADPH-cytochrome P450 reductase ATR2 [J]. Protein expression and purification, 2000, 18(3): 310-5.
- ↑ URBAN P, MIGNOTTE C, KAZMAIER M, et al. Cloning, yeast expression, and characterization of the coupling of two distantly related Arabidopsis thaliana NADPH-cytochrome P450 reductases with P450 CYP73A5 [J]. The Journal of biological chemistry, 1997, 272(31): 19176-86.
- ↑ MIZUTANI M, OHTA D. Two isoforms of NADPH:cytochrome P450 reductase in Arabidopsis thaliana. Gene structure, heterologous expression in insect cells, and differential regulation [J]. Plant physiology, 1998, 116(1): 357-67.
- ↑ https://www.uniprot.org/uniprotkb/Q9FIP9/entry
- ↑ KäRGEL E, MENZEL R, HONECK H, et al. Candida maltosa NADPH-cytochrome P450 reductase: cloning of a full-length cDNA, heterologous expression in Saccharomyces cerevisiae and function of the N-terminal region for membrane anchoring and proliferation of the endoplasmic reticulum [J]. Yeast (Chichester, England), 1996, 12(4): 333-48.
- ↑ FUKUCHI-MIZUTANI M, MIZUTANI M, TANAKA Y, et al. Microsomal electron transfer in higher plants: cloning and heterologous expression of NADH-cytochrome b5 reductase from Arabidopsis [J]. Plant physiology, 1999, 119(1): 353-62.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 907
- 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 907
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 907
- 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 907
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 283
Illegal BsaI.rc site found at 472
Illegal SapI.rc site found at 591
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