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-450(cam), 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 guatte-gaumerine 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.