Dimethylallyl pyrophosphate: tryptophan

Figure 1 Key intermediate and enzymes from the first committed step in ergot alkaloid biosynthesis through the synthesis of lysergic acid. Simple amides of lysergic acid and ergopeptines are covered in Figs 2 and 3, respectively. Abbreviations DMAPP = dimethylallyl pyrophosphate DMAT = dimethylallyl tryptophan AdoMet = S-adenosyl-methionine.

The synthesis of dimethylallyltryptophan (132) by a crude extract of Claviceps purpurea from tryptophan and dimethylallyl pyrophosphate recorded earlier has been reported again recently. In addition to (132), the formation of (133) was observed. (The latter compound, with unspecified stereochemistry around the double bond, has also been isolated from a C. purpurea culture ). It was found further that both (132) and (133) could act as precursors for lysergic acid amides in C. paspali cultures. Both (133) and its (Z)-isomer have been found to act as precursors for elymoclavine (137) but not chanoclavine-I (138) or agro-clavine (136), which are considered to be normal intermediates in elymoclavine biosynthesis.It may be concluded, however, from the combined evidence, that elymoclavine, lysergic acid, and related compounds may normally be formed along an alternative pathway via these allylic hydroxy-compounds. 

It is a monomeric protein of M.W. about 70,000, shows Kj, values for L-tryptophan and dimethylallyl pyrophosphate of 0.067 and 0.2 mM, respectively, and seems to have a relatively low turnover number, about 7 sec . During studies on this enzyme it was observed (13) that agroclavlne and elymoclavine, the terminal alkaloids in the strain used for the isolation of the enzyme, inhibited purified DMAT synthetase. At concentrations of 3 mM ( v<750 mg/1) agroclavlne and elymoclavine inhibited the enzyme 90% and 70%, respectively. The inhibition is of a mixed or uncompetitive type as shown by kinetic analysis with either tryptophan or dimethylallyl pyrophosphate as the variable substrate (Fig. 6). Subsequently, feedback Inhibition by elymoclavine was also demonstrated by GrSger s group (3) for chanoclavlne cyclase and by us for anthranllate synthetase from... 

The biosynthesis of ergot alkaloids is initiated by prenyla-tion of tryptophan with dimethylallyl pyrophosphate (DMAPP) to yield 4-dimethylallyltryptophane (DMAT) (4) (Fig. 35.2) (Floss, 1980). This alkylation step is believed to be rate limiting, and the enzyme that catalyzes this reaction, DMAT synthetase, has been purified more than 60 times (Maier and Groger, 1976 Waller and Dermer, 1981). The enzyme appears to be specific for L-tryptophan and is inhibited by agroclavine (2) and elymoclavine (5) the end products of the reaction. 

Dimethylallyl pyrophosphate L-tryptophan dimethylallyltransferase, chanoclavine-I-cyclase (D 21.2) Agroclavine, elymoclavine, lysergic acid I. R ... 

One example where product inhibition obviously plays a role in living cells is ergoline alkaloid formation in Claviceps (D 21.2). In mycelial mats of Claviceps developing in stationary batch cultures the intracellular concentration of ergoline alkaloids and the activity of dimethylallyl-pyrophosphate L-tryptophan dimethylallyltransferase (DMAT synthase) measurable in homogenates undergo fluctuations Enzyme activity tends to decrease when the intracellular alkaloid concentration is at a maximum. This oscillation may be explained by... 

The ergoline alkaloids are derived from L-tryptophan, dimethylallyl pyrophosphate (D 6) and the methyl group of L-methionine. 

Dimethylallyl pyrophosphate L-tryptophan dimethylallyltransferase (DMAT synthase) ... 

Roquefortine (Fig. 8) is a neurotoxic mold metabolite produced by Penicillium roqueforti that was first isolated by Polonsky et al. 1976 [43]. Roquefortine is also produced by Penicillium crustosum and Penicillium oxalicum. This substance is derived from the oxidative cyclization of tryptophan, histidine, and dimethylallyl pyrophosphate. 

In 1973, Allen demonstrated that the monoprenylated cyclic dipeptide cyclo-L-alanyl-2-(l,l,-dimethylallyl)-L-tryptophan was incorporated into echinulin in cultures of Aspergillus amstelodami [56]. In this study, the reverse prenyiated cyclic dipeptide was doubly labeled with and by treating a partially purified prenyl transferase obtained from Aspergillus amstelodami with cyclo-L-Ala-L-[3- C]-Trp and [l- H]-3-methyl-2-butenyl 1-pyrophosphate (Scheme 33)... 

The findings that mevalonate entered into the ergoline structure in some form of an isoprene unit and that labelled 4-(y,Y-dimethylallyl)pyrophosphate (DMAPP) was incorporated intact into alkaloids (Plieninger et al., 1967) established 4-(Y,y-dimethylallyl)tryptophan (DMAT, 17) as the structural... 

Plieninger, H., Immel, H. and Volkl, A. (1967) Synthese und Einbau eines C- und H-markierten 4-Dimethylallyl-tryptophans und eines C-markierten 4-Dimethylallyl-tryptamins sowie Einbau eines C-markierten Dimethylallyl-pyrophosphates. Liebigs Ann. Chem., 706, 223-229. 

An impressive body of evidence has established that these alkaloids are formed from L-tryptophan (7.25) which condenses at C-4 with dimethylallyl pyrophosphate (7.27), derived in the usual way from (3-7 )-mevalonic acid, to give dimethylallyltryptophan (7.52) [28] this is then modified to give, e.g. elymoclavine (7.55). The main biosynthetic pathway is (7.52) chanoclavine-I (7.55) — agroclavine 7.54) elymoclavine (7.55) lysergic acid (7.55) [27]. 

Dimethylallyltryptophan synthetase (systematic name dimethylallyl-pyrophosphate L-tryptophan dimethylallyltransferase) was isolated by Heinstein et al. (1971). The enzyme was purified to homogeneity from mycelia of Claviceps strain SD 58 (Lee et al., 1976). The enzyme is a single polypeptide chain with molecular weight 73,000. It is activated by... 

McGrath et al. (1973) prepared a cell-free extract (CFE) containing some of the enzymes involved in the biosynthesis of aCA. A mixture of l-[G H] tryptophan and [ 1 - dimethylallyl pyrophosphate was incubated with this... 

CFE at 30°C for 1 hr. aCA isolated from the reaction mixture contained no or while jSCA contained no but 19% of the added It was clear that the CFE contained both an enzyme and a substrate which, along with dimethylallyl pyrophosphate (DMAPP), would produce ]8CA. This cosubstrate was a derivative of tryptophan (Trp), which was isolated and identified as a-acetyl-y-(j5-indolyl)methyltetramic acid (cycloacetoacetyl-L-tryptophanyl, cAATrp), 16. It was also established that this indole, like... 

Fig. 6. j netlcs of inhibition of dimethylallyl-tryptophan synthetase with tryptophan or dlroethyl-allyl pyrophosphate as variable substrate. 

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