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

Angela Rosco, Hubert H. Pauli, Wiebke Priesner, Toni M. Kutchan

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47 Scopus citations

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 μM; K(m) NADPH, 4.2 μM; 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 λ 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 phenol coupling cytochrome P450 of bisbenzylisoquinoline alkaloid biosynthesis in Berberis stolonifera, berbamunine synthase (CYP80A1), in insect cell culture resulted in an alteration of the product profile as compared to that obtained by expression of berbamunine synthase in the absence of plant reductase.

Original languageEnglish
Pages (from-to)369-377
Number of pages9
JournalArchives of Biochemistry and Biophysics
Volume348
Issue number2
DOIs
StatePublished - Dec 15 1997

Keywords

  • Berbamunine synthase
  • Cytochrome c reductase
  • Eschscholzia californica
  • Heterologous expression
  • NADPH-cytochrome P450 reductase
  • Papaver somniferum
  • Saccharomyces cerevisiae
  • Spodoptera frugiperda

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