Molecular cloning, expression, and induction of berberine bridge enzyme, an enzyme essential to the formation of benzophenanthridine alkaloids in the response of plants to pathogenic attack.

Abstract

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.

Reconstitution of a 10-gene pathway for synthesis of the plant alkaloid dihydrosanguinarine in Saccharomyces cerevisiae

“…In plants, the flavoprotein oxidase BBE stereoselectively converts (S)-reticuline to (S)-scoulerine 33 . A truncated version of BBE (PsBBEDN) was cloned with CFS and SPSDN into plasmid pGC994 (Table 1).…”

Phloem-Specific Expression of Tyrosine/Dopa Decarboxylase Genes and the Biosynthesis of Isoquinoline Alkaloids in Opium Poppy.

“…Only the spatial pattern of SS activity has been determined (Gerardy and Zenk, 1993), and it resembles that of TYDC (Facchini and De Luca, 1994). A key step in sanguinarine biosynthesis, the berberine bridge enzyme (BBE), has been cloned and characterized from Eschscholzia californica (Dittrich and Kutchan, 1991). ExpresSion of BBE has been shown to be induced by various elicitors in E. californica cell cultures; however, the tissue-specific expression of BBE has not been investigated.…”

Benzylisoquinoline alkaloid biosynthesis in opium poppy

“…CYPs catalyze a wide variety of reactions ranging from hydroxylations to C-C and C-O couplings and are responsible for the formation of most BIA structural subgroups with the exception of protoberberines. In BIA metabolism, FADOXs have only been implicated in the formation of C-C and C-N bonds, the most noteworthy being the berberine bridge enzyme (BBe), which generates the protoberberine scaffold (Dittrich and Kutchan 1991;Facchini et al 1996;winkler et al 2006). In contrast, FADOX homologs appear to only catalyze the formation of C-O bonds in the formation of non-alkaloid specialized metabolites in other plants (Custers et al 2004;Sirikantaramas et al 2004).…”

“…Protoberberines, protopines and sanguinarine (S)-Scoulerine, the branch-point intermediate leading to protoberberine alkaloids, is formed from (S)-reticuline by BBe (Dittrich and Kutchan 1991;Facchini et al 1996;Kutchan and Dittrich 1995;winkler et al 2006). (S)-Scoulerine is converted via methylenedioxy bridge formation and O-methylation to a variety of protoberberines including (S)-canadine, (S)-stylopine and (S)-sinactine.…”