| Description |
The aromatic prenyltransferase SirD in Leptosphaeria maculans shares sequence homology with fungal indole prenyltransferases and is hypothesized to catalyze the normal O-prenylation of tyrosine in the first committed step in the biosynthesis of the phytotoxin sirodesmin PL. We cloned, expressed, and biochemically characterized SirD and discovered that it was a dimethylallyltyrosine synthase. We synthesized tyrosine and tryptophan derivatives and tested SirD for aromatic substrate promiscuity. NMR studies revealed that SirD catalyzes normal O-, N-, and £-prenylations on tyrosine derivatives and N- and C-prenylations on tryptophan derivatives with normal prenylation at C6 and C7 and reverse prenylation at N1 and C7 of the indole ring, depending on the substituent. The aromatic prenyltransferase dimethylallyltryptophan synthase in Claviceps purpurea catalyzes the normal prenylation of tryptophan at C4 of the indole nucleus in the first committed step of ergot alkaloid biosynthesis. 4-Methyltryptophan is a competitive inhibitor of the enzyme that has been used in kinetic studies. We found that 4-methyltryptophan is an alternate substrate, which gave four products. Similarly, 4- methoxytryptophan and 4-aminotryptophan are alternate substrates. NMR studies revealed that dimethylallyltryptophan synthase catalyzed normal prenylation at N1, C3, C5, and C7 and reverse prenylation at C3 of the indole ring, depending on the substituent. Synthesized aromatic amino acid analogues were also tested with dimethylallyltryptophan synthase. The ability of dimethylallyltyrosine synthase and dimethylallyltryptophan synthase to prenylate six different sites on the indole nucleus, with normal and reverse prenylation at two of the sites, is consistent with a dissociative electrophilic alkylation of the indole ring where orientation of the substrates within the active site and substituent electronic effects determine the position and type of prenylation. |
