Untersuchungen zu einer Methyltransferase und verschiedenen Prenyltransferasen aus dem Sekundärstoffwechsel von Aspergillus-Arten

The main topics of the present dissertation are the biochemical characterisation of a SAM-dependent N-methyltransferase from A. fumigatus and different putative Prenyltransferases from various Aspergillus species. The putative gene fgaMT, consisting of two exons with a size of 272 bp and 748 bp and an intron sequence with a size of 72 bp, was identified in a biosynthetic gene cluster of fumigaclavines in A. fumigatus. Its deduced protein FgaMT comprises 339 amino acids with a molecular mass of 38.1 kDa. The coding region of the gene was successfully amplified from a cDNA library by PCR. After cloning of fgaMT into the expression vector pQE60 the created expression construct pOR15 was transformed into E. coli strain XL1 Blue MRF´. Overexpression of fgaMT was carried out by cultivation at 37 oC, 200 rpm and 1 mM IPTG induction. Soluble FgaMT-His6 was purified to near homogeneity and characterised biochemically. By performing enzyme assays we could prove that FgaMT catalyzes the methylation of 4-dimethylallytryptophan (4-DMAT) in the presence of S-adenosylmethionine (SAM) at the NH2-group. The product of this reaction was identified as 4-dimethylallyl-L-abrine by NMR and mass spectrometry analysis. FgaMT only accepts 4-DMAT but not L-Trp as substrate, which proves that FgaMT is responsible for the second step in the biosynthesis of ergot alkaloids. This enzyme does not require metal ions for its enzymatic activity and shows relatively high substrate specificity towards tryptophan derivatives with a prenyl moiety at position C-4 of the indole ring. 4-DMAT derivatives with modifications at the indole ring were also accepted as substrates. Even 4-methyl-L-tryptophan was accepted by FgaMT. KM values were determined at 0.12 mM for 4-DMAT and 2.4 mM for SAM. The turnover number was 2.0 s-1. FPLC analysis showed that FgaMT acts as a homodimer. The putative prenyltransferase gene NFIA_112230 from Neosartorya fischeri NRRL 181 was successfully amplified by PCR and cloned into expression vector pQE60. This work was started by bachelor student Andreas Schweitzer under my supervision. The expression construct pAS6os was transformed with E. coli XL1 Blue MRF´, the gene was successfully overexpressed and the derived, recombinant protein EAW20699-His6 was isolated. The protein with a molecular mass of about 50 kDa was purified to near homogeneity. To identify its natural substrate a series of enzyme assays with various different substances were carried out and analyzed by HPLC. Until now no prenylation activity could be detected for the recombinant enzyme. Two putative prenyltransferase genes from A. nidulans FGSC A4, named AN9229-PT1 and AN9229-PT2, and the putative prenyltransferase gene ATEG_03092.1 from A. terreus DSM1958 were successfully amplified by Fusion-PCR from Fosmids or gDNA of the appropriate Aspergillus strains, respectively. The amplified genes were cloned into the expression vectors pQE60 and pHis8, and in case of AN9229-PT2, also into pYES2NT/C. The gene sequences were verified by sequencing of the expression constructs. Expression of the genes was successful in the optimized overexpression E. coli strain M15 [pREP4], but the overproduced proteins aggregated to so called inclusion bodies and could not be used for further experiments. In case of AN9229-PT2, it was possible to isolate little amounts of soluble protein by cultivation under mild conditions (low temperature, short cultivation duration and low IPTG concentration) and purification with N-laurylsarcosin, alternatively. Enzyme assays carried out with purified protein as well as with crude extracts in the presence of different possible substrates showed no enzymatic activity until now. Similarily, enzyme assays carried out with crude extracts of AN9229-PT1 and EAU36366 showed the same result.