Joachim Stöckigt

Purification and properties of cinnamyl alcohol dehydrogenase from higher plants involved in lignin biosynthesis

Abstract The purification and characterization of an NADP(H) specific cinnamyl alcohol oxidoreductase is reported. The enzyme, which has been purified 600 ×, shows an absolute specificity for the cinnamyl moiety. The reaction is readily reversible but is strongly inhibited by aldehyde substrates. The enzyme belongs to class A of NAD(P) specific oxidoreductases. The distribution of this activity throughout a wide variety of taxonomically different plant groups as well as plant parts has revealed a possible correlation with lignin biosynthesis.

Three novel enzymes involved in the reduction of ferulic acid to coniferyl alcohol in higher plants: ferulate: Co a ligase, feruloyl-Co a reductase and coniferyl alcohol oxidoreductase

Using a cell-free system from cambial tissue of Sulix alba the reduction of ferulate to coniferyl alcohol has been shown for the first time, unequivocally, to occur in higher plants [l] . This conversion is dependent on ATP, CoA and reduced pyridine nucleotides. A reaction sequence has been postulated on the basis of these experiments involving the intermediate formation of feruloyl-CoA and coniferyl aldehyde which is in accordance with previous assumptions for this mechanism in lignin biosynthesis as recently reviewed [2]. In the present paper, a cell-free preparation of a lignifying higher plant which reduces ferulic acid to coniferyl alcohol is described. The overall reaction is composed of three individual enzymatic steps. Proof is presented that ferulate is first activated by a cinnamate: CoA ligase to feruloyl-CoA which is in turn reduced by NADPH to coniferyl aldehyde. This compound is further reduced to coniferyl alcohol, with NADPH again participating as the reductant.

Isovincoside (strictosidine), the key intermediate in the enzymatic formation of indole alkaloids

Indole alkaloids are among the most complex and diverse low molecular natural products. Their biosynthesis has been investigated by precursor feeding experiments in vivo [l-4] using differentiated Apocynaceae plants. As a result of these studies synthetic vincoside, which was originally assigned the 3 (Y (S) stereochemistry, was found to be incorporated into serpentine and other Corynanthe type alkaloids in good yield while the incorporation of isovincoside with opposite configuration at C-3 was lower by a factor of lo3 [5]. In subsequent experiments the crucial problem of the configuration at C-3 of the alkaloidal secoiridoid glucoside was solved and three independent groups reported vincoside to possess a C-3 (R) /3 hydrogen [6-81. The revision of the absolute C-3 hydrogen stereochemistry to 3 (R) 0 was secured recently by X-ray diffraction experiments [9]. The problem of inversion arose in that vincoside, the C-3 (R) fl epimer, is considered the biosynthetic precursor of the Corynanthe-type alkaloids with 3 a (5’) stereochemistry. Recently, the limitations of in vivo experiments for the elucidation of alkaloid biosynthesis were overcome when Scott and Lee [lo] obtained a crude cell-free system from callus tissue of Cutharunthus roseus which was capable of synthesizing geissoschizine and ajmalicine. These experiments have been extended by us and it was shown that a cell-free preparation from fermenter-grown [ 111 C. roseus cells synthesized ajmalicine, 19.epiajmalicine and tetrahydroalstonine from tryptamine and secologanin in the presence of either NADPH or NADH [ 121. Furthermore, an immediate precursor of these alkaloids was found to accumulate in the absence of reduced pyridine nucleotides which was identified as 20,21 -didehydroajmalicine (cathenamine) with 3 ~(5’) configuration [131* In the present communication we report that using the cell-free system from Cuthurunthus and also an enzyme preparation from Rhuziu strictu cells we were able to isolate the enzymatically catalyzed primary condensation product between tryptamine and secologanin which proved in both cases to be a single product with 3 cr Q configuration. This product is converted by a crude enzyme preparation from C roseus cells to cathenamine and in the presence of reduced nucleotides to ajmalicine, 19.epiajmalicine and tetrahydroalstonine. In contrast to previous assumptions [5,8], the key stone in the intricate pathway which leads to the structurally diverse indole alkaloids is isovincoside (strictosidine) (3 ~1 (s)) and not vincoside (3 p (R)).

Synthesis of ajmalicine and related indole alkaloids by cell free extracts of Catharanthus roseus cell suspension cultures

On the basis of in vivo incorporation experiments, using radioactively labelled precursors, it has been demonstrated in several laboratories [l-4] , that Corynanthe-type indole alkaloids are formed by condensation of tryptamine with a secoiridoid glucoside to yield vincoside. The latter indole glycoside is subsequently transformed into the diverse structures of the indole and dihydro-indole alkaloid series. In order to elucidate the exact mechanism by which these alkaloids are formed and their synthesis regulated, enzymatic studies were necessary. A break through in this aspect occurred recently when Scott and Lee [5] succeeded in obtaining from callus tissue of Clztharanthus roseus, crude cell free system, capable of synthesizing geissoschizine and ajmalicine. For the past two years we have been involved in setting ujp a cell suspension culture system to investigate the conditions for the production of indole alkaloids by cells of C roseus [6] . We now wish to report the enzymatic synthesis of the indole alkaloids; ajmalicine, 19-epiajmalicine and tetrahydroalstonine, and some characteristics of the conditions for the overall enzymatic reaction using this cell culture system.

Reduction of ferulic acid to coniferyl alcohol in a cell free system from a higher plant

Summary A cell free system from cambial tissue of Salix alba has been shown to catalyze the reduction of ferulic acid to coniferyl alcohol. This conversion requires ATP, coenzyme A, and reduced pyridine nucleotides.

Procedure for the enzymatic synthesis and isolation of cinnamoyl-CoA thiolesters using a bacterial system

Abstract A method is described which allows the preparation of pure cinnamoyl-CoA thiolesters in high yields. This procedure utilizes a partially purified cinnamoyl-CoA ligase obtained from a strain of Pseudomonas putida and some properties of this new enzyme are described. Product isolation involves polyamide column chromatography which allows the purification of 50-mg batches of thiolesters. The method is applicable to a range of cinnamic acids, and is particularly suitable in preparing the biologically important CoA esters of p -coumarate, ferulate, and caffeate.

Enzymatic formation of intermediates in the biosynthests of ajmalicine: Strictosidine and cathenamine

Abstract Pre-purified enzymes isolated from Catharanthus roseus suspension cultures synthesize strictosidine and cathenamine from tryptamine and secologanin. Whereas strictosidine showed metabolic activity, cathenamine accumulates during the cell-free incubations in the absence of reduced pyridine nucleotides. In the presence of δ- d -gluconolactone (0.1 M), strictosidine accumulates in a yield of ca 50%. Optimum conditions for its accumulation in crude extracts were found to be at pH 4.1, 0.25 mM tryptamine and 1.25 mM secologinin. Strictosidine synthase is stable for more than 1.5 months at 4°. The optimum conditions for the enzymatic synthesis of cathenamine are 1.54 mM tryptamine and 7.7 mM secologanin at pH 7.5. In the presence of NH 4 + the formation of the latter alkaloid decreases due to the synthesis of unidentified compounds.

Radioimmunoassay for the quantitative determination of scopolamine

Abstract A radioimmunoassay for the determination of pmol amounts of the tropane alkaloid scopolamine has been developed. The assay uses tritiated [N-C3H3]scopolamine of high specific activity (0.67 Ci/mmol) as tracer. The measuring range of the assay extends from 0.5 to 50 ng of scopolamine, and as little as 200 pg may be detected. The antiserum raised against a conjugate of scopolamine-N-β-propionic acid-human serum albumin is highly specific, and neither hyoscyamine, 6-hydroxyhyoscyamine, scopine, tropic acid nor other related alkaloids interfere in the scopolamine determination in crude plant extracts. This assay allows for the first time the rapid, sensitive and precise (CV = 2.5 %) determination of this alkaloid in unpurified extracts of scopolamine-containing plants. The distribution of scopolamine in Datura plants, as well as its diurnal changes in leaf concentrations, has been investigated in detail and a preliminary survey on the variability of scopolamine leaf concentrations in a population of Datura sanguinea plants is given.

Cathenamine, a central intermediate in the cell free biosynthesis of ajmalicine and related indole alkaloids

A compound, which accumulates when tryptamine (I) and secologanin (II) are incubated with an enzyme preparation from Catharanthus roseus cell suspension cultures, was identified as 20,21-didehydroajmalicine (cathenamine)(IVa), and was shown to be a central intermediate in the enzymatic production of ajmalicine (Va),19-epi-ajmalicine (Vb) and tetrahydroalstonine (Vc).

Polyneuridine aldehyde esterase: an unusually specific enzyme involved in the biosynthesis of sarpagine type alkaloids

Polyneuridine aldehyde esterase is a highly substrate specific enzyme which catalyses the conversation of the monoterpenoid C10-unit into the C9–unit at the stage of polyneuridine aldehyde in the biosynthesis of sarpagine type alkaloids.