DMAT synthase

The enzyme responsible for the first step in the biosynthesis of these alkaloids is DMAT synthase. This enzyme, which brings about the condensation of L-tryptophan with DMAPP, has been isolated from C. purpurea cultures. 

We have therefore selectively blocked the first enzyme in the biosynthesis pathway of EA (DMAT-synthase) by 5-fluorotryptophan (Scheme 8). This treatment id not influence other biochemical processes in the living cells of C. purpurea, thus enabling the fructosylation of introduced alkaloids to be performed [27]. 

Scheme 8. Biosynthesis of ergot alkaloids. Selective inhibition of DMAT-synthase in the biosynthesis pathwayby 5-fluorotryptophan blocks synthesis of alkaloids de novo but does not influence other biochemical processes in the cells, e. g., transfructosylating enzymes [27]...

The early steps in the ergot alkaloid biosynthetic pathway are outlined in Fig. 1. The first determinant and rate-limiting step is the prenylation of tryptophan to 4-(y,y-dimethylallyl)tryptophan (DMAT), catalyzed by dimethy-lallyl-diphosphate L-tryptophan dimethylallyltransferase (DMAT synthase EC 2.5.1.34) (Heinstein et al., 1971 Gebler and Poulter, 1992). The prenyl group for the DMAT synthase reaction is provided in the form of dimethylallyl diphosphate (DMAPP), which is derived from mevalonic acid. After the formation of DMAT, the free amino group of this intermediate is N-methylated with a methyl group donated by S-adenosylmethionine (AdoMet). The N-methylated DMAT is then converted into chanoclavine I by closure of the... 

Shibuya et al. (1990) and Gebler and Poulter (1992) provided experimental support that DMAT synthase catalyzes an electrophilic aromatic substitution (Fig. 4) similar to that of other prenyl transferases such as farnesyl diphosphate synthase. A positively charged alkyl intermediate—an allyl carbocation—is... 

Figure 4 Electrophilic aromatic substitution, constituting the most likely mechanism for DMAT synthase. The diphosphate moiety dissociates to generate the allylic carbocation, which then attacks the activated carbon-4 of the indole ring system. Approach of the aromatic C-4 is opposite the diphosphate leaving group, as indicated by experiments with specifically mono-tritiated (T) DMAPP (Shibuya et al., 1990). 

Although several previous reports claimed that the enzyme had been purified, Gebler and Poulter (1992) appear to have been the first to fully characterize the activity of the purified DMAT synthase. The enzyme was purified from Claviceps fusiformis ATCC 26245 [erroneously annotated in type specimen collections as a C. purpurea strain (Pazoutova and Tudzynski, 1999)]. The monomeric size was estimated at 53 kDa, and by gel filtration analysis the native enzyme was determined (at 105 kDa) to be a homodimer. Unlike other prenyltransferases, no metal ion requirement has been noted. However, when assayed in a buffer with 4 mM Ca2+, the purified protein gave a specific activity of 500 nmol/min/mg, essentially the same as with 4 mM Mg2+, but approximately twice that of the measured without added divalent cations and with the chelator EDTA included in the assay buffer. These divalent metal cations eliminated negative cooperativity of substrate binding observed both for dimethylallyl diphosphate and L-tryptophan, indicating that Ca2+ and Mg2+ probably had allosteric effects. In buffer with 4 mM MgCl2 the KM for dimethylallyl diphosphate was 8 jlM, and the KM for L-tryptophan was 12 xM. The enzyme product was authenticated by mass spectrometry, UV spectrometry, and -NMR. 

Interestingly, in limited analyses to date, there is no evidence for clustering of genes encoding DMAT synthase and LPS in Neotyphodium spp. If the Neotyphodium spp. genes are clustered, then their spatial relationships would have to be different from those observed for the genes in the C. purpurea cluster (J. Wang, D. G. Panaccione, C. L. Schardl, unpublished data). 

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... 

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

To rationalize these data, Floss et al. proposed the mechanism shown in Scheme 55 wherein a direct electrophilic aromatic substitution reaction from an enzyme-bound DMAPP ion pair species alkylates C-4 of the tryptophan nucleus [82]. The minor product, where partial loss of stereochemical integrity is sacrificed, was envisioned to occur via rotation around the C-l/C-2 bond of the allylic carbocation species as shown in Scheme 55. Poulter et al. subsequently published a mechanistic study on DMAT synthase that is fully consistent with this interpretation [84]. 

Figure 6 Retention of radioactivity in the pro-R-positions of DMAT after reaction of tryptophan with (IR)- or (15)-[1- H]DMAPP catalysed by DMAT synthase according to Shibuya et al. (1990)...

Reaction mechanism of DMAT formation catalysed by DMAT synthase rotation around the C-l/C-2 bond of the allylic carbocation in a fraction of the molecules prior to bond formation with the indole. These findings characterise the prenyl transfer of DMAT synthase as an electrophilic aromatic substitution, mechanistically similar to the electrophilic alkylation catalysed by farnesyl diphosphate synthase (Song and Poulter, 1994). 

The enzyme responsible for condensation of L-tryptophan and DMAPP is dimethylallylpyrophosphate L-tryptophan dimethylallyl transferase (DMAT synthase). It has been isolated by the group of Floss and these investigators also purified it to homogeneity and performed the first characterisations (Heinstein et al., 1971 Lee et ai, 1976). Initially, the enzyme was reported to be a monomeric... 

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