Light-induced cytochrome P450-dependent enzyme in indole alkaloid biosynthesis: tabersonine 16-hydroxylase.
Vinblastine and vincristine are two medically important bisindole alkaloids from Catharanthus roseus (Madagascar periwinkle). Attempts at production in cell cultures failed because a part of the complex pathway was not active, i.e. from tabersonine to vindoline. It starts with tabersonine 16-hydroxylase (T16H), a cytochrome P450-dependent enzyme. We now show that T16H is induced in the suspension culture by light and we report the cloning of the cDNA. The enzyme was expressed in Escherichia coli as translational fusion with the P450 reductase from C. roseus, and the reaction product was identified by mass spectrometry. The protein (CYP71D12) shares 47-52% identity with other members of the CYP71D subfamily with unknown function. The induction by light was strongly enhanced by a nutritional downshift (transfer into 8% aqueous sucrose). We discuss the possibility that the entire pathway to bisindoles can be expressed in suspension cultures.
Schröder
G
G
Institut für Biologie II, Universität Freiburg, Germany.
Unterbusch
E
E
Kaltenbach
M
M
Schmidt
J
J
Strack
D
D
De Luca
V
V
Schröder
J
J
D000470:Alkaloids / Q000096:biosynthesis* / Q000737:chemistry*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D018076:DNA, Complementary / Q000302:isolation & purification; D004790:Enzyme Induction; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D026121:Indole Alkaloids; D007211:Indoles; D008027:Light; D006899:Mixed Function Oxygenases; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D010944:Plants / Q000201:enzymology* / Q000235:genetics; D011804:Quinolines; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D000470:Alkaloids; D018076:DNA, Complementary; D026121:Indole Alkaloids; D007211:Indoles; D010940:Plant Proteins; D011804:Quinolines; D011993:Recombinant Fusion Proteins; C009373:tabersonine; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C121596:CYP71D12 protein, Catharanthus roseus