A tyrosine O-prenyltransferase catalyses the first pathway-specific step in the biosynthesis of sirodesmin PL

Abstract

A putative prenyltransferase gene sirD has been identified in the gene cluster encoding the biosynthesis of the phytotoxin sirodesmin PL in Leptosphaeria maculans. The gene product was found to comprise 449 aa, with a molecular mass of 51 kDa. In this study, the coding region of sirD was amplified by PCR from cDNA, cloned into pQE70, and overexpressed in Escherichia coli. The overproduced protein was purified to apparent homogeneity, and characterized biochemically. The dimeric recombinant SirD was found to catalyse the O-prenylation of L-Tyr in the presence of dimethylallyl diphosphate; this was demonstrated unequivocally by isolation and structural elucidation of the enzymic product. Therefore, SirD catalyses the first pathway-specific step in the biosynthesis of sirodesmin PL. K m values for L-Tyr and dimethylallyl diphosphate were determined as 0.13 and 0.17 mM, respectively. Interestingly, SirD was found to share significant sequence similarity with indole prenyltransferases, which catalyse prenyl transfer reactions onto different positions of indole rings. In contrast to indole prenyltransferases, which accept indole derivatives, but not Tyr or structures derived thereof, as substrates, SirD also prenylated L-Trp, resulting in the formation of 7-dimethylallyltryptophan. A K m value of 0.23 mM was determined for L-Trp. Turnover numbers of 1.0 and 0.06 S "1 were calculated for L-Tyr and L-Trp, respectively.

Site-directed Mutagenesis Switching a Dimethylallyl Tryptophan Synthase to a Specific Tyrosine C3-Prenylating Enzyme

“…SirD shares a sequence identity of 34% on the amino acid level with 7-DMATS and also accepts L-tryptophan and some of its derivatives as aromatic substrates. It catalyzes mainly a C 7 -prenyla-tion of the indole ring (26,28,29). C 7 -Prenylation of L-tryptophan by tyrosine prenyltransferases was also demonstrated recently with TyrPT from Aspergillus niger (14).…”

“…One example of such enzymes, SirD from Leptosphaeria maculans, is involved in the biosynthesis of sirodesmin PL and responsible for the O-prenylation of the phenolic hydroxyl group in L-tyrosine (26,27). SirD shares a sequence identity of 34% on the amino acid level with 7-DMATS and also accepts L-tryptophan and some of its derivatives as aromatic substrates.…”

“…A few members of the DMATS superfamily catalyze prenylations of non-indole derivatives, and in some cases, O-prenylations (25,26). One example of such enzymes, SirD from Leptosphaeria maculans, is involved in the biosynthesis of sirodesmin PL and responsible for the O-prenylation of the phenolic hydroxyl group in L-tyrosine (26,27).…”

CdpC2PT, a reverse prenyltransferase from Neosartorya fischeri with a distinct substrate preference from known C2-prenyltransferases

“…One subgroup of the prenyltransferases from ascomycetes shares significant sequence similarities with dimethylallyltryptophan synthase (DMATS) in the biosynthesis of ergot alkaloids from Claviceps purpurea (Tudzynski et al, 1999); they are therefore classified as members of the DMATS superfamily. These enzymes catalyse, often by Friedel-Crafts alkylations, the transfer reactions of a prenyl moiety from dimethylallyl diphosphate to diverse aromatic substrates including tyrosine, xanthones, tryptophan and other indole derivatives (Kremer & Li, 2010;Liu et al, 2013;Mundt et al, 2012;Noike et al, 2012;Pockrandt et al, 2012). Indole prenyltransferases of the DMATS superfamily catalyse attachment of prenyl moieties to different positions of the indole ring of tryptophan or derivatives thereof.…”

Regiospecificities and Prenylation Mode Specificities of the Fungal Indole Diterpene Prenyltransferases AtmD and PaxD

“…Moreover, many cyclic dipeptide prenyltransferases have been reported to accept cyclic dipeptide/amino acid derivatives other than their intrinsic substrates (35)(36)(37)(38)(39)(40)(41). For example, FgaPT2, FtmPT1, and 7-DMATS have strict position specificities and essentially introduce DMAPP into the same positions as their intrinsic substrates (the C-4, C-2, and C-7 position, respectively, of the indole moiety).…”