Biochemical Investigations on Microbial Prenyltransferases in the Presence of DMAPP Analogues

Plants, bacteria and fungi provide a plethora of diverse structures derived from the primary as well as the secondary metabolism. Representative substances from secondary metabolite pathways are flavonoids, coumarins, xanthones and indole alkaloids. Although not necessary for the growth and reproduction of the respective organisms, nevertheless these compounds give them an advantage over other organisms in the form of, e.g. attractants or protection agents against natural enemies as well as competitors. The attachment of isoprene units (n C-5) such as dimethylallyl, geranyl or farnesyl moieties to aromatic secondary metabolites as backbones is a further step in the broad diversification of these compounds. Enzymes of the class of prenyltransferases accomplish this prenyl transfer reaction in nature. The prenylated natural products often exhibit strong pharmacological activities in contrast to their substrates. Therefore, prenylated compounds represent interesting targets for drug development. Investigations on alternative alkyl residues transferable via prenyltransferases, i.e. prenyl diphosphate analogues, could have the potential to play an important role in the understanding of the catalytic mechanism of these enzymes as well as in the finding process of new drugs. As the first project in this thesis, the acceptance of four chemically synthesized alkyl and allyl diphosphates in the presence of three L-tryptophan prenyltransferases (FgaPT2, 5 DMATS, 7 DMATS) was elucidated. Retaining the double bond in β position and performing alterations on the methyl groups of the allyl residue led to regular alkylated products and thereby showed successful utilization of DMAPP analogues. However, depending on the DMAPP analogue, the regiospecificity of the applied prenyltransferases was shifted partially or completely. Furthermore, the enzymes did not catalyze the transfer reaction of alkyl diphosphates onto the L tryptophan scaffold, if no double bond was present or if it was relocated to position compared to DMAPP. Subsequently, the behavior of cyclic dipeptide prenyltransferases (AnaPT, CdpNPT, CdpC3PT, FtmPT1, BrePT) towards the unnatural alkyl and allyl donors was examined. The cyclic dipeptide prenyltransferases also used the unsaturated DMAPP analogues resulting in the formation of multiple products. Regardless of the prenylation position and orientation for the prenyl attachment (C 2 or C 3; regular or reverse) in the presence of DMAPP, the enzymatic reactions with both DMAPP analogues, i.e. MAPP and 2-pentenyl-PP, yielded a mixture of C2 reverse as well as C3 reverse alkylated diastereomers in different ratios depending on the donor and used enzyme. After the successful alkylation of L tryptophan and tryptophan-containing cyclic dipeptides utilizing simple unnatural allyl DMAPP analogues, the acceptance of the structurally more complex DMAPP analogue benzyl diphosphate was tested. Preliminary investigations showed the successful usage of this benzyl donor by several L tryptophan and cyclic dipeptide prenyltransferases, whereas FgaPT2 showed the highest activity. FgaPT2 also displayed a remarkable promiscuity using tryptophan analogues as substrates and catalyzing a highly regiospecific C5 benzylation. Consequently, the usage of benzyl diphosphate instead of dimethylallyl diphosphate results in a complete shift of the prenylation position from C 4 to C 5. To complete our findings regarding the acceptance of unnatural alkyl and benzyl analogues by prenyltransferases, several tryptophan C5-, C6- and C7-prenylating enzymes of fungal and bacterial origin have been assayed with the three analogues. Depending on the enzyme used, one to four products could be identified from the incubation mixtures. The predominant products were regular C6-alkylated or C6-benzylated derivatives in all cases. Therefore, for the tested tryptophan C5-, C6- and C7-prenylating enzymes, C-6 seemed to be the preferred position for attachment of the alkyl or benzyl moiety. The results obtained during this thesis show that allyl as well as benzyl analogues of DMAPP are potential alternatives for chemoenzymatic Friedel Crafts alkylations of simple indole derivatives and tryptophan-containing cyclic dipeptides and could be used for the production of alkylated compounds