Eschscholtzia Californica Cell Suspension Cultures Research Articles

Enzymic 10-hydroxylation and 10- O-methylation of dihydrosanguinarine in dihydrochelirubine formation by Eschscholtzia

Two new enzymes involved in benzophenanthridine alkaloid biosynthesis, dihydrosanguinarine-10-hydroxylase and 10-hydroxydihydrosanguinarine-10- O-methyltransferase, have been found in cell-free extracts derived from Eschscholtzia californica cell suspension cultures. The hydroxylase is a microsome-associated, cytochrome P-450-dependent monooxygenase which acts specifically at C-10 of dihydrosanguinarine. The methyltransferase, enriched 60-fold and partially characterized, methylates only the hydroxyl moiety at C-10 to form dihydrochelirubine. Both enzymes were found to be highly substrate specific.

Benzophenanthridine Alkaloids Isolated fromEschscholtzia californicaCell Suspension Cultures Interact with Vasopressin (V1) Receptors

Chelerythrine and sanguinarine, two benzophenanthridine alkaloids, have been isolated from a crude methanolic extract of Eschscholtzia californica cell suspension cultures by successive fractionations. These two molecules exhibited affinity for rat liver vasopressin V1 receptors and are competitive inhibitors of [3H]-vasopressin binding within the micromolar range (Ki). Chelerythrine and sanguinarine represent two of the first non-peptidic structures providing original chemical leads for the design of synthetic vasopressin compounds.

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.

Two methylenedioxy bridge forming cytochrome P-450 dependent enzymes are involved in ( S)-stylopine biosynthesis

The enzymatic synthesis of the alkaloids ( S)-[10-OC 3H 3]scoulerine and ( S)-[3-OC 3H 3]cheilanthifoline is described. A microsomal preparation from Eschscholtzia californica cell suspension cultures in the presence of NADPH and O 2 catalyses the formation of two methylenedioxy bridges in two consecutive steps: ( S)-cheilanthifoline from ( S)-scoulerine and ( S)-stylopine from ( S)-cheilanthifoline. Nandinine was not found to be an intermediate. The release of HO 3H into the aqueous incubation mixture from the radiolabelled substrates served as an assay for both enzyme activities. The enzymes were characterized as to their substrate specificity, cofactor requirements and inhibition by cytochrome P-450 specific inhibitors. Both enzymes were drastically induced (12- and 5-fold) 20 hr after challenging the cell suspension culture with elicitor. The mechanism of enzymic methylenedioxy bridge group formation from an o-methoxyphenol function is discussed.

Elicitor induction and characterization of microsomal protopine-6-hydroxylase, the central enzyme in benzophenanthridine alkaloid biosynthesis

The chemical synthesis of [6- 3H]protopine is described. Microsomal preparations from Eschscholtzia californica cell suspension cultures challenged with a crude elicitor preparation from yeast, catalyse the hydroxylation of [6- 3H]protopine with the concomitant formation of [11- 3H]dihydrosanguinarine and HO 3H. This reaction served as an assay for the enzyme. The hydroxylase reaction is strictly dependent on NADPH as a reducing cofactor and on molecular oxygen. Cytochrome c, inhibitors such as prochloraz and ketoconazole and carbon monoxide inhibit the hydroxylation reaction, suggesting that the enzyme is a cytochrome P-450 linked monooxygenase. The hydroxylase was induced by the yeast elicitor ca eight-fold, 20 hr after challenging the plant cell culture with the yeast carbohydrate, having no effect on the cell dry weight of the culture. The hydroxylase is specifically present in different plant species that produce benzo[c]phenanthridine alkaloids in culture and also respond to induction by the elicitor.

Partial purification and properties of S-adenosyl-l-methionine: (S)-tetrahydroprotoberberinecis-N-methyltransferase from suspension-cultured cells of Eschscholtzia and Corydalis

An enzyme has been found in different species of isoquinoline alkaloid-producing plant cell cultures which specifically N-methylates certain (S)-tetrahydroprotoberberine alkaloids such as (S)-canadine and (S)-stylopine at the expense of S-adenosyl-l-methionine (SAM). It was partially purified (90-fold from Eschscholtzia californica cell suspension cultures and characterized. The enzyme has a pH optimum of 8.9, a temperature optimum at 40° and a Mr of about 78 000 ± 10%. The Km for (S)-canadine was determined to be 6.4,μM, for (S)-stylopine 3.1 μM and for SAM 12,μM. The enzyme is inhibited by S-adenosyl-l-homocysteine (SAH with a Ki of 24 μM.

Partial purification and characterization of dihydrobenzophenanthridine oxidase from Eschscholtzia californica cell suspension cultures

The observation that upon elicitation cell suspension cultures of Eschscholtzia california showed a decrease of dihydromacarpine with a concomittant increase of macarpine led to the discovery of a novel enzyme which catalyzes the oxidation of dihydrobenzophenanthridines in the presence of oxygen. The enzyme was enriched approx. 70-fold. It has a pH-optimum of 7.0, an isoelectric point at pH 8.8, molecular weight of 56 kD and shows a high degree of substrate specificity. The enzyme obviously catalyzes the terminal step in the formation of benzophenanthridine alkaloids containing methylene dioxy substitutions in rings A and D.

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.