Relationship of aromatic prenyltransferases

The enzymes of the DMATS superfamily share very low sequence similarity with the members of the LtxC group and almost no sequence similarity with other known prenyltransferases,e.g.the soluble prenyltransferases of the CloQ/NphB group from bacteria and fungi as well as the membrane-bound prenyltransferases of the UbiA superfamily from bacteria, fungi and plants. Similar to enzymes of the DMATS superfamily, the members of the CloQ/NphB group don’t contain a DDxxD motif. In contrast to those enzymes, the members of the UbiA superfamily contain a NQxxDxxxD motif for prenyl diphosphate binding and are strictly dependent on Mg²⁺or other divalent cations.

Surprisingly, structure analysis revealed that FgaPT2, FtmPT1 and CdpNPT of the DMATS superfamily contain a PT-barrel (Figure 1-19) (Metzgeret al., 2009; Jostet al., 2010; Schuller et al., 2012), which has been only found in the bacterial aromatic prenyltransferases of the CloQ/NphB group (Metzger et al., 2010; Kuzuyama et al., 2005). Therefore, it is proposed that all the proteins from both the DMATS superfamily and the CloQ/NphB group share a common ancestry (Bonitz et al., 2011). It would be interesting to test the acceptance of substrates for the members of the CloQ/NphB group by the prenyltransferases of the DMATS superfamily. An acceptance of hydroxynaphthalenes by a member of the DMATS superfamily was not reported previously. Correspondingly, substrates tyrosine or indole derivatives for the DMATS superfamily were not prenylated by enzymes of the CloQ/NphB group. In vitro assays were carried out in this thesis to test the enzyme activity of prenyltransferases of the DMATS superfamily towards hydroxynaphthalenes. Furthermore, the acceptance of flavonoids by members of the DMATS superfamily was also tested in this thesis.

NphB FtmPT1

Figure 1-19: The structures of NphB (also named Orf2, PDB 1ZB6) and FtmPT1 (PDB 3O2K) in cartoon representation (Kuzuyamaet al., 2005; Jostet al., 2010).

2 Aims of this thesis

The following issues have been addressed in this thesis:

Functional proof of putative indole prenyltransferase genes in Neosartorya and Aspergillus Blast search in GenBank indicated two proteins which showed significant sequence similarity to members of the DMATS superfamily: 5-DMATS from A. clavatus and CdpC3PT from N.

fischeri. 5-DMATS and CdpC3PT showing 52% identity with FgaPT2 and 53% identity with CdpNPT, respectively, indicated similar roles. BrePT from A. versicolor is an orthologue of NotF from Aspergillus sp. MF297-2 and was expected to compensate for low substrate promiscuity of NotF. In order to characterize 5-DMATS, BrePT and CdpC3PT biochemically, the following experiments were carried out:

 Overproduction ofcdpC3PT,5-dmatsandbrePTinE. coli.

 Enzyme assays with CdpC3PT, 5-DMATS and BrePT. Analysis and isolation of enzyme products by HPLC.

 Structure elucidation of enzyme products by NMR and MS.

 Kinetic study of CdpC3PT, 5-DMATS and BrePT reactions.

Chemoenzymatic synthesis of prenylated indole derivatives by indole prenyltransferases of the DMATS superfamily

AnaPT, CdpC3PT and CdpNPT were reported to catalyze reverse prenylation at opposite sides in tryptophan-containing cyclic dipeptides to produce cis-configured prenylated pyrroloindoline diketopiperazines in previous studies (Yin et al., 2010a; Yin et al., 2010b;

Schuller et al., 2012). In this thesis, the stereoselectivity of these three enzymes was demonstrated by analysis of product formation from four cyclo-Trp-Ala and four cyclo-Trp-Pro isomers. In addition, indolocarbazoles, a class of natural products with well known remarkable biological inhibitory effects against protein kinases, were tested for the acceptance by indole prenyltransferases. The following experiments were carried out:

 Synthesis of stereoisomers of several cyclic dipeptides and indolocarbazoles.

 Enzyme assays of cyclic dipeptides and indolocarbazoles with indole prenyltransferases. Analysis and isolation of enzyme products by HPLC.

 Structure elucidation of enzyme products by NMR and MS.

 Kinetic study of the enzyme reactions.

Chemoenzymatic synthesis of prenylated hydroxynaphthalenes and flavonoids by indole prenyltransferases of the DMATS superfamily

As mentioned previously, indole prenyltransferases of the DMATS superfamily showed no sequence similarity and catalytic activity to known aromatic prenyltransferases of the CloQ/NphB group and the UbiA superfamily. However, recent studies indicated that prenyltransferases of the DMATS superfamily share structure similarity with those of the CloQ/NphB group. Therefore, it is interesting to prove if the members of the DMATS superfamily could also accept the substrates for enzymes of the CloQ/NphB group, e.g.

hydroxynaphthalenes. In addition, it is also interesting to know if the members of the DMATS superfamily could catalyze the prenylation of substrates for other known aromatic prenyltransferases, e.g. flavonoids accepted by the members of the UbiA superfamily. In order to investigate the acceptance of hydroxynaphthalenes and flavonoids by the members of the DMATS superfamily, the following experiments were carried out:

 Enzyme assays of hydroxynaphthalenes and flavonoids with indole prenyltransferases. Analysis and isolation of enzyme products by HPLC.

 Structure elucidation of enzyme products by NMR and MS.

 Kinetic study of the enzyme reactions.

Biosynthetic genes other than prenyltransferases involved in the biosynthesis of a prenylated indole alkaloid inA. fumigatus

A gene cluster for the biosynthesis of HAS was identified in A. fumigatus. One NRPS, one putative transporter, two putative C6 transcription factors, one 7-dimethylallyltryptophan synthase (7-DMATS), one putative FAD binding protein, one putative O-methyltransferase (HasC) and one putative cytochrome P450 (HasH) were proposed to be involved in the biosynthesis (Yin et al., 2013b). HasC and HasH were expected to catalyze the O-methylation and N-hydroxylation, respectively. In order to elucidate the biochemical function of HasC and HasH in the biosynthesis of HAS, the following experiments were carried out:

 Cloning ofhasCandhasHinto cloning vector pGEM-T easy.

 Preparing the expression vector ofhasCin pQE60.

 Preparing expression constructs containing hasH: expression vectors of hasH in pESC-URA with or without His6-tag, and the co-expression vector of hasH with a reductase geneNFIA_083630in pESC-URA.

 Overproduction ofhasCinE. coliandhasHinS. cerevisiae.

 Enzyme assays with HasC and HasH.

3 Materials and methods