Molecular biological and biochemical investigations of prenyltransferases from different Aspergillus species
Prenylated aromatic compounds are widely distributed in nature and many of these compounds display interesting biological and pharmaceutical activities. The transfer of prenyl moieties to aromatic substrates is catalysed by aromatic prenyltransferases, and the multitude of possibilities for attac...
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|Summary:||Prenylated aromatic compounds are widely distributed in nature and many of these compounds display interesting biological and pharmaceutical activities. The transfer of prenyl moieties to aromatic substrates is catalysed by aromatic prenyltransferases, and the multitude of possibilities for attachment of these moieties gives rise to a large structural diversity. Investigation of the functions of aromatic prenyltransferases would therefore help to elucidate the biosynthetic pathways of those natural compounds in the producing organism, as well as the applicability of these enzymes for chemoenzymatic synthesis of biologically active compounds for pharmaceutical purposes. Four putative prenyltransferase genes of the dimethylallyltryptophan synthase (DMATS) superfamily from Aspergillus nidulans (A. nidulans) were investigated as one part of this thesis. The genes xptA, xptB, ANID_10289 and ANID_11080 were cloned, and the two putative xanthone prenyltransferase genes xptA and xptB were overexpressed in Escherichia coli (E. coli). Both recombinant prenyltransferases were purified to near homogeneity using affinity chromatography with Ni-NTA and investigated biochemically. In this thesis, the enzyme XptB was confirmed to function as a xanthone prenyltransferase, catalysing a regiospecific Oprenylation of several xanthones. The enzyme products were isolated on high performance liquid chromatography (HPLC) and analysed by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS). Another previously postulated substrate, the benzophenone arugosin H, was not accepted by XptB under the tested conditions. No enzyme activity was detected for the second enzyme XptA with postulated or potential substrates. Reports of the enzyme VrtC from Penicillium aethiopicum (P. aethiopicum) implicated that some members of the DMATS superfamily might not be restricted to the usual prenyl donor dimethylallyl diphosphate (DMAPP, C5-unit). VrtC was shown as the first known member of the DMATS prenyltransferases, which accepts geranyl diphosphate (GPP, C10-unit), but not DMAPP, for prenylation of a naphthacenedione substrate. Therefore, the acceptance of prenyl donors, such as GPP and farnesyl diphosphate (FPP, C15-unit), was investigated in this thesis with the prenyltransferases 7-DMATS and CdpNPT, both from Aspergillus fumigatus (A. fumigatus), as well as CdpC3PT and AnaPT, both from Neosartorya fischeri (N. fischeri). It was found that AnaPT accepted, in addition to DMAPP, also GPP as prenyl donor. Furthermore, a shift of the prenylation position was observed. Instead of a reverse C3α-prenylation with DMAPP, C6- and C7-geranylations of tryptophan-containing cyclic dipeptides with GPP were demonstrated in this thesis. This was the first report of the acceptance of both DMAPP and GPP by a member of the DMATS superfamily. The observed shift of the prenylation position by changing the prenyl donor expands the applicability of AnaPT for the production of prenylated compounds. As last part of this thesis, the putative prenyltransferase genes AO090102000322, AO090701000600 and AO090020000527 from Aspergillus oryzae (A. oryzae) were cloned. The recombinant protein BAE61387, encoded by AO090102000322, was successfully overproduced and purified from E. coli. Analysis of neighbouring genes in the genome of A. oryzae revealed no backbone genes for the biosynthesis of a possible natural substrate of BAE61387. The enzyme BAE61387 was therefore incubated with a large number of compounds that are known substrates of enzymes from the DMATS superfamily. Activity was clearly observed in incubation mixtures with hydroxynaphthalenes. Interestingly, these unnatural substrates, which are accepted by several members of this enzyme class, were accepted by BAE61387 in the presence of all three prenyl donors DMAPP, GPP and FPP. The acceptance of all these prenyl donors for regiospecific C-prenylations of hydroxynaphthalenes has never been shown for a member of this enzyme superfamily prior to this work.|