Overproduction, purification and characterization of FtmPT1, a brevianamide F prenyltransferase from Aspergillus fumigatus

Abstract

A putative prenyltransferase gene, ftmPT1, was identified in the genome sequence of Aspergillus fumigatus. ftmPT1 was cloned and expressed in Escherichia coli, and the protein FtmPT1 was purified to near homogeneity and characterized biochemically. This enzyme was found to catalyse the prenylation of cyclo-L-Trp-L-Pro (brevianamide F) at the C-2 position of the indole nucleus. FtmPT1 is a soluble monomeric protein, which does not contain the usual prenyl diphosphate binding site (N/D)DXXD found in most prenyltransferases, and which does not require divalent metal ions for its enzymic activity. K m values for brevianamide F and dimethylallyl diphosphate were determined as 55 and 74 mM, respectively. The turnover number was 5?57 s "1 . FtmPT1 showed a high substrate specificity towards dimethylallyl diphosphate, but accepted different tryptophan-containing cyclic dipeptides. Together with dimethylallyltryptophan synthase of ergot alkaloid biosynthesis, FtmPT1 belongs to a new group of prenyltransferases with aromatic substrates. INTRODUCTIONTrans-prenyltransferases (Bohlmann et al., 1998;Liang et al., 2002;Sacchettini & Poulter, 1997) are involved in the biosynthesis of terpenoids from C-5 units. These enzymes usually contain one or more prenyl diphosphate binding motifs, (N/D)DXXD, in their sequences (Bohlmann et al., 1998;Liang et al., 2002;Sacchettini & Poulter, 1997). Their enzymic reaction depends strictly on the presence of metal ions such as Mg 2+ or Mn 2+ (Bohlmann et al., 1998;Liang et al., 2002). A special group of prenyltransferases catalyses the attachment of a prenyl moiety to an aromatic nucleus. These enzymes show similar sequence motifs and metal ion requirements to those of the trans-prenyltransferases. Examples of this group are the prenyltransferases involved in the biosynthesis of the primary metabolites ubiquinone (Turunen et al., 2004), menaquinone (Suvarna et al., 1998), tocopherol (Schledz et al., 2001) and plastoquinone (Collakova & DellaPenna, 2001), and the prenyltransferase involved in the formation of the plant secondary metabolite shikonin (Yazaki et al., 2002). All these enzymes are membrane-bound proteins.In contrast, the dimethylallyltryptophan synthase (DMATS) that catalyses the prenylation of tryptophan at position C-4 of the indole nucleus during ergot alkaloid biosynthesis in the fungus Claviceps has been found to be a soluble protein and is active in a metal-free buffer containing EDTA (Cress et al., 1981;Lee et al., 1976;Tsai et al., 1995; Tudzynski et al., 1999). Recently, two further soluble prenyltransferases with aromatic substrates, which are active in the absence of metal ions and contain no (N/D)DXXD motifs, CloQ involved in the biosynthesis of clorobiocin from Streptomyces roseochromogenes (Pojer et al., 2003) and LtxC involved in the biosynthesis of lyngbyatoxins from Lyngbya majuscula (Edwards & Gerwick 2004), have been identified. Very recently, we identified an orthologue of DMATS, FgaPT2, in the genome sequence of Aspergillus fumigatus (Unsöld & Li, 2005). Inte...

Genome mining and biosynthesis of fumitremorgin-type alkaloids in ascomycetes

“…A gene cluster for the biosynthesis of fumitremorgin-type alkaloids was initially identified by our group in the genome of A. fumigatus Af293. 27 This strain was the first sequenced Aspergillus species and its genome sequence was released to the public in 2003. By blasting the genome sequence with cpd1, a prenyltransferase gene coding for a 4-dimethylallyltryptophan synthase in the biosynthesis of ergot alkaloids in Claviceps purpurea, 28 a segment of 25 kbp on chromosome 8 was identified as a putative biosynthetic gene cluster for fumitremorgin B.…”

“…By blasting the genome sequence with cpd1, a prenyltransferase gene coding for a 4-dimethylallyltryptophan synthase in the biosynthesis of ergot alkaloids in Claviceps purpurea, 28 a segment of 25 kbp on chromosome 8 was identified as a putative biosynthetic gene cluster for fumitremorgin B. 27 The identified cluster was proposed to comprise 10 genes. 27 Later on, orthologous clusters were also identified in A. fumigatus A1163 and N. fischeri NRRL181.…”

“…27 The identified cluster was proposed to comprise 10 genes. 27 Later on, orthologous clusters were also identified in A. fumigatus A1163 and N. fischeri NRRL181. 29,30 Comparison of these clusters revealed the presence of all of the 10 genes in both strains.…”

“…27,31,32,35 In total, functions of seven genes have been proven experimentally. On the basis of the obtained bioinformatic, molecular biological and biochemical results, the following biosynthetic pathway has been postulated ( Figure 3).…”

“…1 The biosynthesis of fumitremorgin-type alkaloids begins by condensation of the two amino acids L-tryptophan and L-proline to brevianamide F, catalyzed by the non-ribosomal peptide synthetase FtmPS (also termed FtmA), which was proven by heterologous overexpression of the NRPS gene ftmPS in Aspergillus and identification of the accumulated product brevianamide F. 33 Brevianamide F is then prenylated by the prenyltransferase FtmPT1/FtmB in the presence of dimethylallyl diphosphate, resulting in the formation of tryprostatin B, as demonstrated by using the recombinant FtmPT1. 27 By gene inactivation in A. fumigatus BM939 and heterologous expression in S. cerevisiae, Kato et al 31 showed that the three cytochrome P450 enzymes, FtmP450-1/FtmC, FtmP450-2/FtmE and FtmP450-3/FtmG, are responsible for the conversion of tryprostatin B (2) to 6-hydroxytryprostatin B, tryprostatin A (3) to fumitremorgin C (8) and fumitremorgin C (8) to 12,13-dihydroxyfumitremorgin C, respectively. Using recombinant enzymes, we have demonstrated that FtmPT2/FtmH catalyzes the prenylation of 12,13-dihydroxyfumitremorgin C in the presence of dimethylallyl diphosphate, resulting in the formation of fumitremorgin B, 35 which is then converted to verruculogen by FtmOx1/FtmF by inserting an endoperoxide bond between the two prenyl moieties.…”