Purification and Properties of Strictosidine Synthase, the Key Enzyme in Indole Alkaloid Formation

Abstract

A new enzyme, strictosidine synthase, which catalyzes the synthesis of 3-a(S)-strictosidine from tryptamine and secologanin was isolated from the soluble protein extract of Catharanthus roseus cell suspension cultures and was purified approximately 50-fold by ammonium sulfate fractionation, column chromatography on DEAE-cellulose, Ultrogel AcA34 and isoelectric focusing. The apparent molecular weight of the enzyme was 34000. The pH optimum was 6.8, apparent K, values for tryptamine and secologanin were 2.3 mM and 3.4 mM respectively for the enzyme to synthesize strictosidine. Strictosidine synthase shows high substrate specificity. No apparent cofactor requirement could be demonstrated. Of several enzyme inhibitors tested, only p-chloromercuribenzoate inhibited the enzyme. The enzyme was relatively stable and could be stored at -20 "C for periods of up to 1 year without appreciable loss of catalytic activity. The enzyme was demonstrated to occur in suspension cultures of 15 different species belonging to 9 different genera of the indole-alkaloidproducing subfamily Plumerioideae of the Apocynaceae family.This enzyme is responsible for the synthesis of strictosidine the key intermediate in the formation of the majority of monoterpenoid indole alkaloids occurring in the plant kingdom.The indole alkaloids, of which about 1200 are known to date [l], make up the largest class of alkaloids. They comprise such chemically diverse structures as ajmaline, gelsemine, quinine, strychnine, vinblastine etc. By feeding experiments in vivo, it was shown that most of the monoterpenoid alkaloids are formed through the condensation of the iridoid secologanin with tryptamine (reviewed 121). Recently the key enzyme, catalyzing the stereospecific condensation of tryptamine with secologanin to yield the alkaloidal glucoside strictosidine with 3 -4 9 configuration, was discovered [3 -51 and some properties of the enzyme from crude extracts of cell cultures of alkaloid-producing Apocynaceae plants were preliminarily reported for the first time [6]. Strictosidine is the universal precursor for the multitude of monoterpenoid indole alkaloids both of the 3-c( as well as of the 3-1) series [7,8]. This enzyme catalyzes a PictetSpengler-type reaction between the aldehyde function of secologanin and the amino group of tryptamin as shown in Fig. 1. This cyclisation suggests a Schiff-base formation followed by an electrophilic attack of C-2 of the indole ring. During this attack the hydrogen atom of C-2 of the indole was eliminated, which formed the basis for a convenient and sensitive assay of this enzyme using the ring [2-'H]tryptamine as substrate and analysing the H03H formed for radioactivity [6].In extension of our previous work [6], we have now purified strictosidine synthase from Catharanthus roseus (= Vinca rosea) cell suspension cultures. This

Strictosidine Synthase: Mechanism of a Pictet−Spengler Catalyzing Enzyme

“…Strictosidine synthase, originally isolated from Catharanthus roseus and R. serpentina almost 30 years ago, 15,16 has been the subject of numerous steady-state kinetic analyses (for two examples, see refs 17,18 ). K m values have been reported for both tryptamine 1 (4 μM) and secologanin 2 (40 μM), and values previously reported from our laboratory match literature values.…”

“…In this analysis, we make the assumption that the K m values for the enzyme do not change significantly over the pH range measured and that the ternary complex is still accessible to proton transfer with the solution. Although the pH dependence of strictosidine synthase has previously been measured, 16,18 the pK a values corresponding to these changes in enzyme activity have not been reported. We therefore measured the rate of strictosidine synthase in a series of buffers at fixed ionic strength under saturating substrate concentrations.…”

Molecular Architecture of Strictosidine Glucosidase: The Gateway to the Biosynthesis of the Monoterpenoid Indole Alkaloid Family

“…Although broad biosynthetic knowledge is the prerequisite for metabolic engineering (Kutchan, 1995), the characterization of the enzymes responsible for the biosynthesis of these alkaloids is still somewhat preliminary, and details of the enzymatic formation have been obtained for only very few pathways. For instance, several enzymes were detected in cell suspensions of Catharanthus roseus G. Don, participating in the biosynthesis of ajmalicine and its isomers (Stö ckigt, 1979;Treimer and Zenk, 1979). In addition, some enzymes involved in the formation of the alkaloid vindoline in plant seedlings and cell culture of C. roseus have been previously well characterized (De Luca et al, 1985;Fahn et al, 1985;St-Pierre et al, 1998;StPierre and De Luca, 2000).…”

The cDNA clone for strictosidine synthase from Rauvolfia serpentina DNA sequence determination and expression in Escherichia coli

“…INTRODUCTION The alkaloidal glucoside, strictosidine, was recognized in 1968 as a biosynthetic precursor of monoterpenoid indole alkaloids [I]. There was, however, much confusion about the stereochemistry of this first intermediate in the pathway [2,3] which was only clarified after the enzyme responsible for the stereospecific condensation of tryptamine with secologanin was discovered [4,5] and characterized [6,7]. This precursor of over 1800 indole alkaloids, some of high commercial value [8], has been unequivocally shown to possess a ICY configuration 191.…”

“…This precursor of over 1800 indole alkaloids, some of high commercial value [8], has been unequivocally shown to possess a ICY configuration 191. The enzyme catalyzing the formation of strictosidine was named stric- tosidine synthase [5,6]. A convenient assay has been developed to monitor this reaction [6] and the enzyme from Rauvolfia serpentina has been purified to homogeneity [lo].…”