Molekularbiologische und biochemische Untersuchungen zu Prenyltransferasen und NRPS-ähnlichen Enzymen aus Ascomyceten

Microorganisms developed the capability to produce bioactive secondary metabolites to ensure their own viability and get evolutionary advantages. These metabolites can be used as antibiotics or immunosuppressive drugs, but also as lead structures for the development of new drugs like HIV protease inhibitors or insulin mimetics. Therefore, microorganisms are an important source of potentially active pharmaceutical ingredients. Significant progress has been achieved in the past decades in the research on the biosynthesis of these compounds. On the one hand, nonribosomal peptide synthetases and polyketide synthases are common enzymes involved in the biosynthesis of the backbones of numerous metabolites and their module-based structure represents a target for manipulations with the means of synthetic biology. On the other hand, secondary metabolites can be further modified by tailoring enzymes like prenyltransferases which often leads to a higher biological activity. It is of particular importance to investigate these structures and to understand their functions in order to make use of those enzymes. Many prenylated bisindolylbenzoquinones belonging to prenylated indole alkaloids have been isolated from ascomycetes, but biochemical information has been available only for the biosynthesis of terrequinone A in Aspergillus nidulans prior to this thesis. In this thesis the putative prenyltransferase genes astPT and ATEG_00702 (EAU39348) from Aspergillus terreus were expressed heterologously in Escherichia coli and subsequently purified to near homogeneity. The recombinant protein AstPT catalysed the N- and C-prenylation of asterriquinone D in the presence of dimethylallyldiphosphate (DMAPP). The enzyme products, two mono- and two diprenylated compounds, were isolated and their structure was elucidated by NMR spectroscopy and mass spectrometry. As other prenyltransferases from the DMATS-superfamily, AstPT was not dependent on the presence of metal ions for its catalytic activity. The reaction apparently followed Michaelis-Menten kinetics and the Michaelis constant was determined at 463 µM with a turnover number at 0.16 s-1 for AQ D and at 33.5 µM and 0.02 s 1 for DMAPP, respectively. Special features of AstPT were the introduction of prenyl moieties at two distinct sites of the substrate and its high specificity towards its aromatic substrate. It accepted neither amino acids nor cyclic dipeptides, which are typical substrates of prenyltransferases of the DMATS-superfamily. However, it accepted four hydroxyxanthone derivatives as substrates and catalysed O-prenylations in the presence of DMAPP but also from geranyl- (GPP) and farnesyldiphosphate (FPP). This phenomenon has only been observed for one enzyme from this group before. The best accepted xanthone derivative had a Michaelis constant at 17.3 µM, but the turnover number for AQ D was 160 times higher than for this hydroxyxanthone. EAU39348 was not able to convert AQ D. Using didemethylasterriquinone D (DDAQ D) as aromatic substrate, the formation of several products was observed. The slow conversion of AQ D by AstPT and the exclusive acceptance of DDAQ D by EAU39348 may indicate that the production of DDAQ D derivatives is preferred in Aspergillus terreus. The reason for this might be the stronger biological activities of DDAQ D derivatives in comparison to AQ D derivatives. A third putative prenyltransferase gene, CHGG_03684, was cloned from Chaetomium globosum and overproduction was tested in different expression systems. Unfortunately, no soluble protein was obtained under these conditions. An attempt of renaturation via dialysis was made after solubilisation of inclusion bodies with urea, but no activity was observable after incubation with AQ D and DMAPP. Furthermore, four putative NRPS-like genes from Aspergillus terreus and Chaetomium globosum were amplified via PCR. ATEG_02004, ATEG_03563 and CHGG_03687 were cloned into the expression vector pJW24 containing pyrG as a selection marker for subsequent transformations. The construct with ATEG_02004 was successfully trans-ferred into Aspergillus niger AB 1.13 and the genomic integration was confirmed via PCR. The function of ATEG_02004 could not be identified by investigation of the produced secondary metabolites. In addition, the importance of several amino acid residues in FtmPT1 was studied in this thesis to support the proposed reaction mechanism, which is based on the enzyme structure. The involvement of glutamate 102 in the stabilization of the indole nitrogen and its role as proton acceptor during catalysis was shown in activity assays after site-directed mutagenesis and overproduction of the proteins. Exchange of glycine 115 for threonine led to a shift of the prenylation position from C 2 to C 3 and the introduction of a hexahydropyrroloindole. This was observed by using the natural substrate brevianamide F as well as different cyclic dipeptides. These results revealed the essential role of G115 in the prenylation of FtmPT1. Mutation of the corresponding amino acid in CdpC3PT did not have any effect on the prenylation position and simply decreased the activity. The exchange of tyrosine 205 for phenylalanine increased the acceptance of six hydroxynaphthaline derivatives.