Molecular cloning and heterologous expression of a cDNA encoding berbamunine synthase, a C--O phenol-coupling cytochrome P450 from the higher plant Berberis stolonifera.
A cDNA encoding a cytochrome P450-dependent oxidase, berbamunine synthase (EC 1.1.3.34; CYP80), from cell suspension cultures of the higher plant Berberis stolonifera Koehne and Wolf (barberry) has been isolated and heterologously expressed in functional form in insect cell culture using a baculovirus-based expression system. This cytochrome P450-dependent enzyme is unusual in that it catalyzes the regio- and stereoselective formation of a C--O phenol couple in bisbenzylisoquinoline alkaloid biosynthesis without concomitant incorporation of activated oxygen into the product. Consistent with the function of an oxidase rather than a monooxygenase, an essential glycine residue in the distal helix, which forms the oxygen-binding pocket in the well-studied bacterial enzyme P-450cam, is replaced by proline at the equivalent position in berbamunine synthase. This oxidase was accumulated in an active form in insect cell microsomes and accepted electrons from the endogenous NADPH-cytochrome P450 reductase. The heterologously expressed enzyme oxidatively couples either two molecules of (R)-N-methylcoclaurine to form the (R,R) dimer guattegaumerine or one molecule each of (R)- and (S)-N-methylcoclaurine to form the (R,S) dimer berbamunine. The ratio of the two bisbenzylisoquinolines formed could be altered by reductase source or by varying the enantiomer composition of the substrates.[1]References
- Molecular cloning and heterologous expression of a cDNA encoding berbamunine synthase, a C--O phenol-coupling cytochrome P450 from the higher plant Berberis stolonifera. Kraus, P.F., Kutchan, T.M. Proc. Natl. Acad. Sci. U.S.A. (1995) [Pubmed]
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