Molecular cloning and characterization of CYP719, a methylenedioxy bridge-forming enzyme that belongs to a novel P450 family, from cultured Coptis japonica cells.
Two cytochrome P450 (P450) cDNAs involved in the biosynthesis of berberine, an antimicrobial benzylisoquinoline alkaloid, were isolated from cultured Coptis japonica cells and characterized. A sequence analysis showed that one C. japonica P450 (designated CYP719) belonged to a novel P450 family. Further, heterologous expression in yeast confirmed that it had the same activity as a methylenedioxy bridge-forming enzyme (canadine synthase), which catalyzes the conversion of (S)-tetrahydrocolumbamine ((S)-THC) to (S)-tetrahydroberberine ((S)-THB, (S)-canadine). The other P450 (designated CYP80B2) showed high homology to California poppy (S)-N-methylcoclaurine-3'-hydroxylase (CYP80B1), which converts (S)-N-methylcoclaurine to (S)-3'-hydroxy-N-methylcoclaurine. Recombinant CYP719 showed typical P450 properties as well as high substrate affinity and specificity for (S)-THC. (S)Scoulerine was not a substrate of CYP719, indicating that some other P450, e.g. (S)-cheilanthifoline synthase, is needed in (S)-stylopine biosynthesis. All of the berberine biosynthetic genes, including CYP719 and CYP80B2, were highly expressed in selected cultured C. japonica cells and moderately expressed in root, which suggests coordinated regulation of the expression of biosynthetic genes.
Ikezawa
Nobuhiro
N
Division of Integrated Life Sciences, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan.
Tanaka
Masaru
M
Nagayoshi
Masanori
M
Shinkyo
Raku
R
Sakaki
Toshiyuki
T
Inouye
Kuniyo
K
Sato
Fumihiko
F
D000429:Alcohol Oxidoreductases / Q000737:chemistry; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001599:Berberine / Q000737:chemistry; D001600:Berberine Alkaloids / Q000737:chemistry; D002248:Carbon Monoxide / Q000737:chemistry; D003001:Cloning, Molecular; D039323:Coptis / Q000201:enzymology*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics*; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015723:Gene Library; D005822:Genetic Vectors; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009249:NADP; D010100:Oxygen / Q000378:metabolism; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012313:RNA / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D017422:Sequence Analysis, DNA; D013997:Time Factors
D000596:Amino Acids; D001600:Berberine Alkaloids; D018076:DNA, Complementary; D011994:Recombinant Proteins; C092555:cytochrome P-450 CYP80A1 (Berberis stolonifera); D001599:Berberine; D009249:NADP; D012313:RNA; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System; D000429:Alcohol Oxidoreductases; C092892:tetrahydrocolumbamine; D010100:Oxygen