Cloning and heterologous expression of NADPH-cytochrome P450 reductases from the Papaveraceae.
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 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.
Rosco
A
A
Laboratorium für Molekulare Biologie, Universität München, Germany.
Pauli
H H
HH
Priesner
W
W
Kutchan
T M
TM
D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D000470:Alkaloids / Q000096:biosynthesis / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010207:Papaver / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry; D010946:Plants, Medicinal; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera / Q000235:genetics
D000470:Alkaloids; D010446:Peptide Fragments; D011994:Recombinant Proteins; C092555:cytochrome P-450 CYP80A1 (Berberis stolonifera); D003577:Cytochrome P-450 Enzyme System; D000429:Alcohol Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase