Biochemische Untersuchungen von Prenyltransferasen aus verschiedenen Ascomyceten

Aspergillus fumigatus, an opportunistic pathogenic fungus, is the major reason for invasive aspergillosis and constitute also as an allergenic origin. Despite of the importance of the pathogenity of this fungus, only little is known about the background of its pathogenity. A. fumigatus produces a series of secondary metabolites which can be alone or together responsible for the pathogenity. For example, this fungus produces gliotoxin, an epipolythiodixopiperazine, which is in vitro characterised as a potent agent for cell death. For this reason it was speculated, that this mycotoxin might be involved in the pathogenesis of A. fumigatus. It is still necessary to know more about the genetic information and biosynthesis of further secondary metabolites, which might function as virulence factors. The biosynthetic gene cluster of gliotoxin has been identified in A. fumigatus. In the genome sequence of A. fumigatus Af293, an additional putative biosynthetic gene cluster containing three genes with significant sequence similarity to gliC, gliP und gliM of the gliotoxin cluster, as well as to sirC, sirP and sirM of the sirodesmin cluster could be identified. Sirodesmin is also an epipolythiodixopiperazine derivative, which is produced by Leptosphaeria maculans. Sirodesmin is implicated in the phytopathogenity of this fungus. The putative gene cluster from A. fumigatus contains in total eigth open reading frames. The end product of this cluster is still unknown. One gene in this cluster, Afu3g12930 (termed hereafter 7-dmats), which encodes for a putative prenyltransferase, could be identified. The coding region of this gene was cloned, overexpressed in Escherichia coli and afterwards the His6-fusion protein was purified. The enzyme was found to catalyse the prenylation of various aromatic substances whith the highest conversation rate for L-tryptophan. This indicated that 7-DMATS functions as a dimethylallyltryptophansynthase. Structural elucidation of the enzymatic product revealed that the prenyl moiety was attached at position C-7 of the indole moiety. As mentioned above, 7-DMATS accepted various aromatic substrates, including different tryptophan-containing cyclic dipeptides and different indole derivatives. 24 simple indole derivatives were tested and all of the substances, with an exception of 7-methyltryptophan, were accepted and converted to their prenylated products. Structural elucidation of these prenylated products proved the prenylation at position C-7 and demonstrated that 7-DMATS catalyses regiospecifically the prenylation at position C-7 of the indole moiety. Eight different indole derivatives could be prenylated with conversion rates between 56 and 100 % under optimised conditions. Based on the broad substrate specificity and the high conversation rate, 7-DMATS can be used as an effective tool for chemoenzymatic synthesis of prenylated indole derivatives. 7-DMATS is an interesting enzyme not only due its potential as a tool for chemoenzymatic synthesis, but also its catalytic promiscuity.