Berberine Bridge Enzyme Research Articles

Heterologous expression of the plant proteins strictosidine synthase and berberine bridge enzyme in insect cell culture

The heterologous expression of cDNAs encoding the alkaloid biosynthetic enzymes, strictosidine synthase [EC 4.3.3.2] from Rauvoifia serpentina and the berberine bridge enzyme [( S)-reticuline: oxygen oxidoreductase (methylene-bridge-forming), EC 1.5.3.9] from Eschscholtzia californica, has been achieved in a cell culture (Sf9) of the fall army worm, Spodoptera frugiperda, using a baculovirus-based expression system. The expression resulted in the overproduction of each plant enzyme in a catalytically active form. The maximal production attained was 4 mg purified, active enzyme per litre cell culture for both the strictosidine synthase and berberine bridge enzymes.

12-Oxo-Phytodienoic acid induces accumulation of berberine bridge enzyme transcript in a manner analogous to methyl jasmonate

The induction kinetics for berberine bridge enzyme [(S)-reticuline : oxygen oxidoreductase (methylenebridge-forming), EC 1.5.3.9] encoding poly (A)+ RNA were determined for Eschscholtzia californica (California poppy) cell suspension cultures treated with either a yeast cell wall elicitor preparation methyl jasmonate or 12-oxo-phytodienoic acid. It was found that all three inducers produce similar patterns of transcript accumulation.

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.

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 californica, and partial amino acid sequences have been determined. A cDNA, isolated from a Agt11 cDNA bank of elicited E. californica 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. californica 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.

Elicitation of benzophenanthridine alkaloid synthesis in Eschscholtzia cell cultures

Quaternary benzophenanthridine alkaloids (sanguinarine, chelerythrine, chelirubine, chelilutine and macarpine) are specifically induced by cell wall components of Penicillium and Saccharomyces in a colorless strain of Eschscholtzia californica cell suspension cultures. Classical elicitors such as the Phytophthora megasperma elicitor are inactive. The alkaloid synthesis is, however, strongly induced by certain polypeptide antibiotics. Out of 190 tested plant species the yeast elicitor provoked benzophenanthridine synthesis in 13 cultures. One of the branch point enzymes, namely the berberine bridge enzyme, catalysing the formation of (S)-scoulerine from (S)-reticuline, is strongly stimulated during the elicitation process. These results clearly demonstrate the induction of the benzophenanthridine biosynthetic pathway by microbial elicitors.

Intracellular compartmentation of two enzymes of berberine biosynthesis in plant cell cultures

Out of the eight enzymes involved in the biosynthesis of the isoquinoline alkaloid berberine, at least, two enzymes, berberine bridge enzyme and (S)-tetrahydroprotoberberine oxidase, are exclusively located in a vesicle with a specific gravity of ϱ=1.14 g·cm(-3) as shown by direct enzymatic assay as well as immunoelectrophoresis. Electronmicroscopic examination of the enzyme-containing particulate preparation from Berberis wilsoniae var. subcaulialata cultured cells demonstrated that it is composed mainly of membranous vesicles. The protein composition of this preparation reveals the presence of only about 20 separable proteins, of which two major ones are berberine bridge enzyme and (S)-tetrahydroprotoberberine oxidase. Incubation of these vesicles with the substrate (S)-reticuline in the presence and absence of S-adenosyl-L-methionine leads to the formation of a red product which was identified as dehydroscoulerine. If the cytoplasmic enzyme S-adenosyl-L-methionine:(S)-scoulerine-9-O-methyltransferase is added to the vesicle preparation in the presence of (S)-reticuline and S-adenosyl-L-methionine, not dehydroscoulerine but columbamine, the immediate precursor of berberine is formed. Some of the quaternary alkaloids are located inside the vesicles; fusion of these vesicles leads to vacuoles containing the quaternary alkaloids. These vesicles are the first highly specific and unique compartment serving only alkaloid biosynthesis; they are found in members of four different plant families and in cell cultures as well as in differentiated tissue.

Purification and characterization of the berberine bridge enzyme from berberis beaniana cell cultures

The berberine bridge-forming enzyme (BBE) has been found in 66 samples taken from differentiated plants and from cell suspension cultures. It was purified 450-fold from Berberis beaniana cell cultures by gel-filtration, DEAE and phenyl-Sepharose chromatography, electrophoresis and isoclectric focusing. The enzyme was shown to be homogeneous by gel electrophoresis ( M r = 52 kD ± 4). The enzyme, which requires the presence of oxygen, catalyses the conversion of the ( S)-enantiomers of reticuline, protosinomenine and laudanosoline to the corresponding ( S)-tetrahydroprotoberberines and released stoichiometric amounts of H 2O 2. Within the cells the enzyme is located in a particle with the density p = 1.14 g/ml.

Zur Rolle eines Reticulin-umwandelnden Enzyms (Berberin-Brücken-Enzym) im pflanzlichen Benzylisochinolin-Stoffwechsel

Investigations on the role of the reticuline converting enzyme for the biosynthesis of tetrahydro-protoberberine (THPB) alkaloids gave the following results: 1. The enzyme activity in cell suspension cultures of Macleaya microcarpa increases before onset of the biosynthesis of THPB alkaloids. 2. The reticuline converting enzyme and THPB metabolism show a concomitant presence in different plant species. 3. The enzyme converts not only (±)-reticuline but also (±)-laudanidine into THPB alkaloids. Obviously, it is specialized in the conversion of certain benzyltetrahydroisoquinoline alkaloids. An essential prerequisite for the enzymatic reaction seems to be the hydroxyl group at the C-atom 3′ of the given skeleton. — According to these findings we propose to replace the name “reticuline converting enzyme” by “berberine bridge enzyme”.