Structure-based protein engineering and biochemical investigations of fungal prenyltransferases for targeted production of novel prenylated indole alkaloids

Prenyltransferases (PTs) catalyze the transfer of one or more prenyl units with chain lengths of n x C5 (n = 1, 2 etc.) to diverse acceptors. Prenylations are essential in both the primary and secondary metabolism of all organisms. PTs are often involved in the biosynthetic pathways of secondary met...

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Bibliographic Details
Main Author: Mai, Peter
Contributors: Li, Shu-Ming (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2019
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Summary:Prenyltransferases (PTs) catalyze the transfer of one or more prenyl units with chain lengths of n x C5 (n = 1, 2 etc.) to diverse acceptors. Prenylations are essential in both the primary and secondary metabolism of all organisms. PTs are often involved in the biosynthetic pathways of secondary metabolites (SMs). Many indole alkaloids (IAs) from Ascomycota are such SMs generated by diverse enzymes and PTs of the dimethylallyl tryptophan synthase (DMATS) superfamily with L-tryptophan as a precursor. These PTs mostly use the prenyl donor dimethylallyl diphosphate (DMAPP) to catalyze metal ion-independently and regiospecific Friedel-Crafts alkylation at the indole ring. The increased lipophilicity of prenylated substances is often associated with improved bioactivity. Prenylated tryptophan-containing cyclic dipeptides (CDPs) with a 2,5 diketopiperazine ring and their derivatives are such representative structures with significant biological and pharmacological activities. Echinulin and derivatives thereof possess antiviral, antitumor and neuroprotective activities. Fumitremorgin C is a potential inhibitor of the breast cancer resistance protein. Tryprostatin A and B are known as microtubule inhibitors. The demand for new bioactive substances such as antibiotics or cancer therapeutics prompted in this thesis the targeted manipulation of PTs on the basis of protein structure data. The obtained mutants were then used for the production of new prenylated IAs. Furthermore, pharmacologically relevant substances were produced by the combination of chemical and enzymatic synthesis. In this thesis, five tryptophan PTs, FgaPT2, 5-DMATS, 5-DMATSSc, 6-DMATSSa and 7-DMATS, were investigated for their potential to prenylate the tripeptide derivative ardeemin fumiquinazoline (FQ) and its stereoisomers. Ardeemin FQ is the precursor of the reverse C3-prenylated ardeemin and 5-N-acetylardeemin. These substances block the MultiDrug Resistance (MDR) export pump in cancer cells, thereby increasing the potency of cancer therapeutics such as Vinca alkaloids in the cell. This thesis demonstrates that PTs from other biosynthetic gene clusters are also able to prenylate ardeemin FQ. The chemically synthesized stereoisomers could also be converted by these PTs, which has not been reported prior to this study. Here, the two stereo centers of these substrates impact the activity and regioselectivity of these enzymes. 18 new prenylated tripeptide derivatives were obtained. Since the tryptophan PTs catalyze only prenylations on the indole’s benzene ring of the tripeptide derivatives, five further PTs were investigated in a cooperation project with Lindsay Coby. BrePT, FtmPT1, CdpNPT, CdpC3PT and AnaPT are cyclic dipeptide PTs that prenylate their natural or best-accepted substrates at the pyrrole ring of the indole. A high conversion rate with ardeemin FQ has been determined for BrePT, FtmPT1 and CdpNPT, whereas CdpC3PT and AnaPT show less acceptance. The enantiomer was poorly converted by all enzymes. These results also show that the stereochemistry of the substrates affects their acceptance by the cyclic dipeptide PTs. 8 new pyrrole-ring prenylated tripeptide derivatives were produced. From both projects, 26 new obtained substances are available for further studies on their bioactivity. Another focus of this thesis is the structure-based protein manipulation through targeted mutation to generate PTs with novel catalytic properties and their application for the production of new prenylated IAs. For this purpose, FgaPT2, which catalyzes regular C4-prenylation of L-tryptophan, was used as a model for mutational experiments. The targeted combinational mutation of two amino acids (K174 and R244) has produced several FgaPT2 double mutants that catalyze reverse C3-prenylation of CDPs with high product yields. This work is a cooperation with Liujuan Zheng. In another study, targeted mutations were performed also on FgaPT2, which is a DMAPP-specific PT, in order to expand the prenyl donor specificity. Molecular modeling identified methionine 328 as potential key amino acid residue for the acceptance of the prenyl donor. The FgaPT2_M328G mutant shows a high turnover with GPP and low acceptance with FPP. An enzymatic geranylation or farnesylation of tryptophan as free amino acid was not described prior to this study. The decrease in DMAPP-acceptance by different FgaPT2_M328X mutants indicate that, in addition to size, the polarity of the amino acid also contributes to prenyl donor selectivity. The mutants with an amino acid smaller than methionine (FgaPT2_M328X; X = C, A, T, S, G, V, N) show high rates of GPP-utilization. The mutants FgaPT2_M328X (X = C, S, G, A) also possess FPP-activity. Due to their polar properties, high GPP- and high DMAPP-acceptance was observed by FgaPT2_M328X (X = C, T, V, N) mutants. To increase FPP-utilization, L263 and Y398 were identified as potential amino acids. The generation of the mutants FgaPT2_L263A_M328A and FgaPT2_L263A_M328A_Y398F led to a further increase in the FPP-utilization. In this study, 21 mutants were produced and 7 mutants can be used as geranyltransferases for chemoenzymatic syntheses of new IAs. In a cooperation study with Ge Liao, five additional DMATSs were selected for mutation experiments to achieve GPP-activity. The C2-PT FtmPT1 catalyzes a regular, BrePT and CdpC2PT a reverse transfer of the prenyl residue from DMAPP to the C-2 position of CDPs. The C3-PTs CdpNPT and CdpC3PT attach the prenyl residue to the C-3 position of CDPs in a reverse manner. Based on sequence alignments and molecular modeling, M364 in FtmPT1, I337 in BrePT, T351 in CdpC2PT, M349 in CdpNPT and F335 in CdpC3PT were identified to be responsible for prenyl donor selectivity and thus were replaced by glycine via site-directed mutagenesis. The generated mutants FtmPT1_M364G, BrePT_I337G, CdpNPT_M349G and CdpC3PT_F335G show clear acceptance of GPP compared to their wildtype. The CdpC2PT wildtype has already accepted GPP. The mutant CdpC2PT_T351G, however, displays a different geranylation pattern. 42 geranylated derivatives could be obtained from 15 CDPs. For cyclo-L-Trp-L-Trp the geranyl moiety of GPP could be transferred to almost all possible positions.
Physical Description:328 Pages