Abstract
Of thirteen steps in the biosynthesis of berberine, the antimicrobial tetrahydrobenzylisoquinoline alkaloid, two are catalyzed by cytochromes P450 (P450). Interestingly, one of them catalyzes a cyclization of an ortho-methoxyphenol, which is unique and a difficult reaction to mimic in organic chemistry. We report here the isolation of this novel methylenedioxy ring-forming P450 cDNA from cultured Coptis japonica cells.
Two P450 cDNAs were isolated from a cDNA library of high berberine-producing Coptis japonica, using PCR fragments amplified with primers designed for the conserved region of P450. Their gene products were designated CYP80B2 and CYP719 by P450 nomenclature committee. Recombinant CYP80B2 produced in yeast showed (S)-N-methylcoclaurine-3'-hydroxylase activity as CYP80B1 isolated from California poppy had. Recombinant CYP719 showed the methylenedioxy ring-forming activity, which converts (S)-tetrahydrocolumbamine to (S)-tetrahydroberberine. Further enzymological analysis showed that CYP719 had high substrate affinity and specificity, indicating that it is specific to berberine biosynthesis.
