No abstract

No abstract

No abstract

No abstract

No abstract

A novel oxidase, catalyzing in the presence of oxygen the removal of four hydrogen atoms from a number of tetrahydroprotoberberines with simultaneous production of 1 mol H 2 0 2 and H 2 0 each, has been discovered and purified to homogeneity from Berberis wilsoniae cell cultures. This enzyme, (9-tetrahydroprotoberberine oxidase, exhibited strict specificity for the (8-enantiomer of tetrahydroprotoberberines and 1 -benzylisoquinoline alkaloids, a pH optimum at 8.9, a molecular mass of 105 kDa and consisted of two subunits each of 53 kDa and covalently bound flavin. The K , values for (8-scoulerine and (8-norreticuline were 25 pM and 150 pM respectively. Concentration of the end-products, either protoberberines or H202, greater than 0.5 mM caused severe enzyme inhibition. This catalyst was responsible for the conversion of (3-tetrahydrocolumbamine to the key intermediate, columbamine, in the metabolic pathway leading to berberine, jatrorrhizine and palmatine.Berberine and jatrorrhizine are members of the large class of protoberberine alkaloids, containing an oxidized C-ring system, which are widely distributed in nature [l]. Both compounds are produced in large amounts in plant cell cultures [2, 31 and a commercial process for the production of the medicinally useful alkaloid, berberine, by Coptis japonica cell suspension culture has been established [4]. Protoberberines containing a quaternary nitrogen atom are formed from the central branch-point intermediate of the benzylisoquinoline pathway, (9-reticuline [l]. This molecule is able to undergo many different transformations including the formation of the so-called berberine bridge in the tetrahydroprotoberberine molecule, which is derived from the N-CH3 group of reticuline [5, 61. The enzyme that catalyzes this transformation has recently been discovered [7] and characterized [8]. The first tetrahydroprotoberberine formed, (9-scoulerine, is subsequently methylated at the OH group in position 9 with Sadenosylmethionine as methyl donor [9]. (8-Tetrahydrocolumbamine thus formed is then aromatized in ring C to form columbamine [lo], a branch-point intermediate for the late steps in the protoberberine biosynthesis [ll], and also gives rise to berberine by formation of a methylenedioxy ring at ring A (Fig. 1).The oxidation of (3-tetrahydrocolumbamine to columbamine, which implies the oxidation of a chiral (8-tetrahydroprotoberberine to a non-chiral protoberberine system, is catalyzed by (8-tetrahydroprotoberberine oxidase [lo]. In this paper we report on the purification and some properties of (S)-tetrahydroprotoberberine oxidase, an enzyme which occupies a key position in protoberberine biosynthesis, from suspension cells of Berberis wilsoniae var subcaulialata Schneid. A preliminary account of this work hasCorrespondence to

From the dried roots of Rauwolfia serpentina were isolated five new indole alkaloids, N(b)-methylajmaline (1), N(b)-methylisoajmaline (2), 3-hydroxysarpagine (3), yohimbinic acid (4), isorauhimbinic acid (5), a new iridoid glucoside, 7-epiloganin (6), and a new sucrose derivative, 6'-O-(3,4,5-trimethoxybenzoyl)glomeratose A (7), together with 20 known compounds. The structures of the new compounds were determined by spectroscopic and chemical means. The inhibitory activities of the selected alkaloids on topoisomerase I and II and their cytotoxicity against the human promyelocytic leukemia (HL-60) cell lines were assessed.

A new enzyme, (S)-norlaudanosoline synthase, which catalyses the synthesis of (S)-norlaudanosoline from dopamine and 3,4-dihydroxyphenylacetaldehyde was isolated from the soluble protein extract of ESCHSCHOLTZIA TENUIFOLIA cell suspension cultures and purified approximately 40-fold. The apparent molecular weight of the enzyme is 15 500 Dalton. The pH optimum is 7.8, temperature optimum 40 degrees C, apparent K (M) values for dopamine and dihydroxyphenyl-acetaldehyde are 1.5 mM and 0.7 mM respectively. The synthase shows high substrate specificity in that only the phenylacetaldehydes are transformed but not the phenylpyruvates. No apparent cofactor requirement could be demonstrated. By means of isoelectric focusing and disc-gel electrophoresis evidence was obtained for the existence of four norlaudanosoline synthase isoenzymes, none of which catalyses the reaction of dopamine with 3,4-dihydroxyphenylpyruvate. These enzymes are responsible for the synthesis of (S)-norlaudanosoline, the key intermediate in the formation of isoquinoline alkaloids occurring in the plant kingdom.