Cloning and heterologous expression of NADPH-cytochrome P450 reductases from the Papaveraceae

Archives of Biochemistry and Biophysics
A RoscoT M Kutchan

Abstract

Cytochrome P450 reductase was purified to homogeneity from cell suspension cultures of the opium poppy Papaver somniferum, the enzyme was characterized (K(m) cytochrome c, 8.3 microM; K(m) NADPH, 4.2 microM; pH optimum, 8.0; M(r), 80 kDa), and the amino acid sequence of internal peptides was determined. Partial cDNA clones from P. somniferum and from Eschscholzia californica (California poppy) were then generated using the polymerase chain reaction and were used as hybridization probes to isolate full-length cDNAs. The Papaver and Eschscholzia cytochrome P450 reductases are 63% identical at the nucleotide level and 69% identical at the amino acid level. SDS-PAGE of the purified native P. somniferum enzyme as well as genomic DNA gel blot analysis indicate that two cytochrome P450 reductase isoforms are present in each species. This evidence is also supported by translation of nucleotide sequences obtained from the PCR-generated partial cDNAs and the full-length cDNAs isolated from lambda libraries. The Papaver and Eschscholzia cytochrome P450 reductases were functionally expressed in the yeast Saccharomyces cerevisiae and in the insect cell culture Spodoptera frugiperda Sf9. Coexpression of cytochrome P450 reductase with the C-O...Continue Reading

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Citations

Sep 29, 1998·The Plant Journal : for Cell and Molecular Biology·W M Chou, T M Kutchan
Jul 2, 2009·The Journal of Biological Chemistry·Andreas GesellToni M Kutchan
May 5, 2001·Annual Review of Plant Physiology and Plant Molecular Biology·Peter J Facchini
Sep 9, 2004·Proceedings of the National Academy of Sciences of the United States of America·Marion WeidToni M Kutchan
Nov 26, 2009·Phytochemistry·Kenneth Jensen, Birger Lindberg Møller
May 6, 2015·The Plant Journal : for Cell and Molecular Biology·Megan M AugustinToni M Kutchan
Jan 15, 2013·Biochemical and Biophysical Research Communications·Guillaume A W Beaudoin, Peter J Facchini
May 20, 2015·Metabolic Engineering·Isis J Trenchard, Christina D Smolke
Jul 5, 2007·Plant Cell Reports·Sonia Gómez-GaleraPaul Christou

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