The berberine bridge enzyme [(S)-reticuline: oxygen oxidoreductase (methylene-bridge-forming), EC 1.5.3.9] is a vesicular plant enzyme that catalyzes the formation of the berberine bridgehead carbon of (S)-scoulerine from the N-methyl carbon of (S)-reticuline in a specific, unparalleled reaction along the biosynthetic pathway that leads to benzophenanthridine alkaloids. Cytotoxic benzophenanthridine alkaloids are accumulated in certain species of Papaveraceae and Fumariaceae in response to pathogenic attack and, therefore, function as phytoalexins. The berberine bridge enzyme has been purified to homogeneity from elicited cell-suspension cultures of Eschscholtzia calafornica, and partial amino acid sequences have been determined. A cDNA, isolated from a Agtll cDNA bank of elicited E. calzfornica cell-suspension cultures, coded for an open reading frame of 538 amino acids. The first 22 amino acids constitute the putative signal peptide. The mature protein has a Mr of 57,352, excluding carbohydrate. The berberine bridge enzyme was heterologously expressed in a catalytically active form in Saccharomyces cerevisiae. Southern hybridization with genomic DNA suggests that there is only one gene for the enzyme in the E. californica genome. Hybridized RNA blots from elicited E. calafornica cell-suspension cultures revealed a rapid and transient increase in poly(A)+ RNA levels that preceded both the increase in enzyme activity and the accumulation of benzophenanthridine alkaloids, emphasizing the integral role of the berberine bridge enzyme in the plant response to pathogens.

The berberine bridge enzyme ((S)-reticuline:oxygen oxidoreductase (methylene-bridge-forming), EC 1.5.3.9) catalyzes the oxidative cyclization of the N-methyl moiety of (S)-reticuline into the berberine bridge carbon, C-8, of (S)-scoulerine. This is a reaction that has neither an equivalent in organic chemistry nor a parallel in nature. The uniqueness of this catalytic reaction prompted an in depth study that began with the isolation of the cDNA encoding the berberine bridge enzyme followed by the overexpression of this cDNA in insect cell culture. The heterologously expressed enzyme has herein been shown to contain covalently attached FAD in a molar ratio of cofactor to protein of 1:1.03. Sitedirected mutagenesis and laser desorption time-of-flight mass spectrometry suggest that the site of covalent attachment is at His-104. The holoenzyme exhibited absorbance maxima at 380 and 442 nm and a fluorescence emission maximum at 628 nm (310 nm excitation). Enzymic transformation of a series of (S)-reticuline derivatives modified with respect to the stereochemistry at C-1 or in the aromatic ring substitution suggests that ring closure proceeds in two steps: formation of the methylene iminium ion and subsequent ring closure via an ionic mechanism.

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The pcnta,;¥clic biosynthetic precursor ofjaimonic a=id. 12-oxo-phytodicnoic acid, was found to induce =ynth~is of t~ major flavonoid, apih=, in ¢¢11 sufpenfion cultur¢s oF Pe#ro#¢linum cri=pum. The accumulation of apiin was p~e_~_dexl by an incr',.am in the rclativ¢ levels of pol~ (A)" RNAs that ¢od¢ for the flavonoid biosynthetic enzymes phenylalanine ammonia Ixa~, 4-¢oumarate:CoA iigat¢ and ¢hal¢one synthaf¢, Poly (A)" RNAt reached maximal levels at approximately 4-6 h after the addition of elicitor while flavonoids continued to accumulate in the ¢ultur ,,t for at leatt 6 day~, 12.Oxo.phytodi=noic add is the first p=ntacy¢lic pr¢=u~or in the jasmoni¢ acid biosynthetic chain which functions as a signal transd~r for phxtoalexin induction.

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