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Prenylated compounds are widely distributed in all living organisms and often posses biological activities differing from their non-prenylated precursors. Transfer reactions of an isoprene moiety from a prenyl donor onto an acceptor molecule, e. g. a terpenoid, a serine residue of a protein or an aromatic molecule, are catalyzed by prenyltransferases. Aromatic prenyltransferases are responsible for prenylation of aromatic compounds and found as membrane-bound or soluble enzymes in bacteria, fungi and plants. They contribute to the diversity of primary and secondary metabolites. During the last decades, many prenylated natural products were isolated from different microorganisms. These natural products include aurachins, a group of quinoline alkaloids from myxobacteria. In this thesis the gene auaA from the putative biosynthetic gene cluster of aurachins in the myxobacterium Stigmatella aurantiaca Sg a15 was heterologously expressed in E. coli and the gene product was subsequently characterized biochemically. AuaA was unequivocally demonstrated to function as a farnesyltransferase catalyzing the prenylation of 2-methyl-4-hydroxyquinoline at position C3 of the quinoline ring. The enzymatic reaction of AuaA followed apparently Michaelis-Menten-kinetics and was strictly dependent on the presence of divalent metal ions. KM-values were determined for 2-methyl-4-hydroxyquinoline and FPP at 0.27 mM and 0.043 mM, respectively. The maximum reaction velocity Vmax was determined at 2.21 nmol/min*mg. Like other aromatic prenyltransferases AuaA showed also flexibility towards aromatic substrates. In addition to its natural substrate 2-methyl-4 hydroxyquinoline, this membrane-bound enzyme accepted also six other 4-hydroxyquinolines and low conversion was even observed for two of six tested flavonoids. In contrast to the promiscuity towards aromatic substrates, AuaA was found to be relatively specific for FPP. GPP was accepted only with a relative activity of 3.3 % in comparison to FPP.
Selected amino acid residues in the aspartate-rich motifs, which are characteristic for membrane-bound aromatic prenyltransferases, were investigated in AuaA by site-directed mutagenesis. By mutation of the conserved aspartate residues, the importance of these residues was confirmed. Mutation of the arginine residue in the first aspartate-rich motif (NRxxDxxxD) revealed that the amino acid at this position is not directly involved in the AuaA reaction, which is in contrast to the results obtained for LePGT1, another prenyltransferase from this group.
The enzymes FgaPT2, FtmPT1 und 7-DMATS from Aspergillus fumigatus belong to aromatic prenyltransferases of the DMATS-family and catalyze the transfer of a prenyl moiety onto an indole ring. In contrast to membrane-bound aromatic prenyltransferases, they do not contain aspartate-rich motifs for binding of prenyl diphosphate via divalent metal ions and their activities are independent of the presence of metal ions. Therefore it was speculated that basic amino acid residues in prenyltransferases of the DMATS-family coordinate the prenyl diphosphate residue. To investigate the importance of three conserved basic amino acid residues for the catalytic activites, site-directed mutagenesis experiments were carried out with FgaPT2, FtmPT1 and 7-DMATS. The results indicated that two lysine residues are involved in the prenyl transfer catalyzed by these enzymes. Mutation of arginine, the third chosen amino acid residue, led only in FgaPT2 to a strong decrease of enzyme activity, indicating that the corresponding arginine residues in FtmPT1 and 7-DMATS are not directly involved in the prenylation reactions.
After solution of the crystal structure of FgaPT2 and analyzes of the enzyme complex with L-tryptophan and DMASPP, a substrate analogue to dimethylallyl diphosphate (DMAPP), a reaction mechanism was postulated. In this mechanism the amino acid residues E89, R100 and K174 should play essential roles. In order to verify this hypothesis, mutagenesis experiments of the suggested residues were carried out and the effects of the mutations on the enzyme activity were determined. Mutation of E89 and R100 confirmed the essential roles of these residues for the catalysis of FgaPT2. In contrast, substitution of K174 by glutamine did not support the importance of this residue for the postulated reaction mechanism.