Biochemical investigations on bacterial and fungal prenyltransferases

Prenylated natural products are widely distributed in nature and demonstratean amazing varietyof structures and promising biological activities, which are usually distinct from their non-prenylated precursors. Therefore, they are intensively studied by researchers from different disciplines. These includeinvestigationson their biosynthesis as well as on the involved key enzymes such as prenyltransferases, which contribute significantly to the structural diversity and biological activity. Prenyltransferases catalyze the transfer reactionsof prenyl moietiesfrom different prenyl donors to various aliphatic or aromatic acceptors.This thesis focuses on the prenyltransferases from the dimethylallyltryptophan synthase (DMATS) superfamily. A new member of this superfamily TyrPT was characterized biochemically in vitro. The responsible geneAn13g01840had been identified in the genome sequence ofA.niger and cloned into pET28a. In this thesis, TyrPT was found to catalyze theO-prenylation of tyrosine as well as C7-prenylation of tryptophan. It was further compared with the known tyrosine O-prenyltransferase SirD and tryptophan C7-prenyltransferase 7 DMATS toward a series of tyrosine and tryptophan derivatives. TyrPT exhibited a broader substrate spectrum and significantly higher catalytic activity for several substrates than the other two enzymes.Kinetic parameters of TyrPT reactions withten substrateswere determined.This studynot only providesanew enzyme,but also enhances the relationshipsbetween tyrosine O- andtryptophan C-prenyltransferases. Further studiesdemonstrated that tryptophan prenyltransferases FgaPT2 and 7-DMATS also accepted tyrosine and its derivatives as substrates and catalyzed a unique C3- and O-prenylation, respectively. A mechanism ofthe FgaPT2 reaction with tyrosine was proposed based on the molecular modeling results with the available crystal structure of FgaPT2.Based on this hypothesis, sixteen mutated FgaPT2 derivatives were tested with tryptophan and tyrosine as substrates. The mutant K174F demonstrated much higher catalytic ability toward tyrosine than FgaPT2 and showed almost no activity toward tryptophan. Therefore, tryptophan C4-prenyltransferase was switched to a specific tyrosine C3-prenyltransferase by site-directed mutagenesis. This strategy was also used for enhancing thecatalytic activity of FgaPT2as a C4-prenylating enzyme of cyclic dipeptides. Thirteen mutated FgaPT2 with much higher catalytic activities and different substrate preferences toward cyclic dipeptides were obtained as new biocatalysts. Inspired by the results above, the L-tyrosineanalogs,L-o- andL-m-tyrosine, were tested with the tryptophan prenyltransferases FgaPT2, 5-DMATS, 6-DMATSSv and 7 DMATS as well as the tyrosine prenyltransferases SirD and TyrPT. Surprisingly,SirD hardly accepted thesetyrosine analogs. In contrast,tryptophan prenyltransferases generally demonstrated higher catalytic activities toward these two substrates. Product isolation and structure elucidation proved C5-prenylated o-tyrosine as unique product of these enzymes, and both C4- andC6-prenylated derivativeswere identified as the products of the FgaPT2 reaction with L-m-tyrosine.These results revealed that chemical category of the aromatic nucleus was not the essential featureforthe acceptance of a substrate by tryptophan or tyrosine prenyltransferases. It strongly depends on thesubstitution positions of the functional groups on the aromatic nucleus, which directly interact with the enzyme active sites. Aforementioned results exhibitfairly the broad substrate spectra of DMATS enzymes. However, the tryptophan prenyltransferases usually accepted cyclic dipeptides only at high protein concentrations.Consequently, tryptophan was a verypoor substrate for cyclic dipeptide prenyltransferases. In this thesis, the substrate promiscuity of indole prenyltransferases including tryptophan and tryptophan-containing cyclic dipeptide prenyltransferases was further expanded by their acceptance of the synthesized unnatural cyclo-L-homotryptophan-D-valine with one additional C-atom between the indole and the diketopiperazine rings.This compound was well accepted by all the tested prenyltransferases including three tryptophan and five cyclic dipeptide prenyltransferases. Seven prenylated products with one prenyl moiety at each position of the indole nucleus were isolated from the enzyme assays.This was the firstreport on the production of seven monoprenylated productsfrom one substrate by one-step reactions. The results presented in this thesis demonstrate that prenyltransferases of theDMATS superfamily are a rich source of biocatalysts, which could play an important role forproduction of prenylated compounds.