Molecular biological and biochemical investigations on the biosynthetic enzymes of prenylated indole alkaloids from fungi

Prenylated indole alkaloids are widely distributed in plants, fungi and bacteria, especially in the family of Clavicipitaceae and Trichocomaceae of Ascomycota, and commonly exhibit interesting biological and pharmaceutical activities. In the biosynthetic pathway of prenylated indole alkaloids, prenylation catalyzed by prenyltransferases contributes significantly to the large structure diversity of these compounds in nature. Investigation on indole prenyltransferases would help to understand the construction of prenylated indole alkaloids in nature and also be useful for structural modification of indole derivatives and other substances to produce analogues of prenylated derivatives. Three indole prenyltransferases belonging to the dimethylallyltryptophan synthase (DMATS) superfamily were biochemically identified and characterized in vitro, including CdpC3PT from Neosartorya fischeri (N. fischeri), BrePT from Aspergillus versicolor (A. versicolor) and 5-DMATS from Aspergillus clavatus (A. clavatus). The responsible genes cdpC3PT and brePT were cloned into expression vector and heterologously expressed in Escherichia coli (E. coli). These works were carried out by Dr. Wen-Bing Yin, Suqin Yin and Qing Wang, respectively. In this thesis, CdpC3PT was confirmed to catalyze the formation of C3-prenylated products with a characteristic 6/5/5/6-fused tetracyclic ring system from tryptophan-containing cyclic dipeptides in one-step reaction. The NotF homologue BrePT showed much higher flexibility towards its aromatic substrates than NotF, and was proven to catalyze the highly regiospecific reverse prenylation at C-2 of the indole nucleus. The cloning of 5-dmats was carried out by Yan Liu. Functional proof of this gene was provided within this thesis by heterologous expression in E. coli and subsequent structure elucidation of enzyme products by mass spectrometry (MS) and nuclear magnetic resonance (NMR) analyses. 5-DMATS established high regiospecific activity to catalyze C5-prenylation on indole derivatives. Given the importance of prenylation in structure diversity and bioactivity enhancement, CdpC3PT, BrePT, 5-DMATS and other known prenyltransferases of the DMATS superfamily were applied for the chemoenzymatic synthesis of prenylated compounds. By using AnaPT, CdpC3PT and CdpNPT, eight and six stereoisomers of cis-configured prenylated pyrroloindoline diketopiperazines from cyclo-Trp-Ala and cyclo-Trp-Pro isomers were produced, respectively. The stereospecificity of AnaPT and CdpC3PT depended mainly on the configuration of tryptophanyl moiety in cyclo-Trp-Ala and cyclo-Trp-Pro isomers, while CdpNPT showed lower stereoselectivity, but higher conversion ability towards most tested substrates. 5-DMATS and FgaPT2 from Aspergillus were used for chemoenzymatic synthesis of prenylated indolocarbazoles. Reconstitution of enzyme activity of 5-DMATS and FgaPT2 in vitro revealed that they catalyzed regiospecific prenylation of indolocarbazoles at the para-position of the indole N-atom. This is the first report for prenylated indolocarbazoles. Subsequently, some indole prenyltransferases of the DMATS superfamily were found to accept also hydroxynaphthalenes and flavonoids, which were substrates for enzymes from the CloQ/NphB group and the UbiA superfamily, respectively. Nine prenylated flavonoids and twenty prenylated hydroxynaphthalenes have been isolated, and their structures were elucidated by MS and NMR analyses. It has been shown that, for an accepted hydroxynaphthalene, different enzymes produced usually the same major prenylated product, i.e. with a regular C-prenyl moiety at para- or ortho-position to a hydroxyl group. For flavonoids accepted by 7-DMATS, C-6 between two hydroxyl groups was the favorable prenylation position. The Michaelis-Menten constants (KM) and turnover numbers (kcat) of some prenyltransferases towards selected hydroxynaphthalenes are comparable to those obtained by using indole derivatives. In addition to indole prenyltransferases, other genes in the biosynthetic cluster of prenylated indole alkaloids were also investigated. A putative O-methyltransferase gene hasC and a putative cytochrome P450 gene hasH involved in the biosynthesis of hexadehydroastechrome (HAS) in Aspergillus fumigatus (A. fumigatus) were cloned into pQE60 and pESC-URA, respectively. Soluble His6-HasC was successfully overproduced in E. coli SG13009 and purified to near homogeneity by Ni-NTA. Constructs for co-expression with the reductase gene NFIA_083630 from N. fischeri in pESC-URA and for expression as His6-tagged protein in pESC-URA were also prepared for the putative cytochrome P450 gene hasH