ABSTRACT
Previous studies showed that verruculogen is the end product of a biosynthetic gene cluster for fumitremorgin-type alkaloids in Aspergillus fumigatus and Neosartorya fischeri. In this study, we isolated fumitremorgin A from N. fischeri. This led to the identification of the responsible gene, ftmPT3, for O-prenylation of an aliphatic hydroxy group in verruculogen. This gene was found at a different location in the genome of N. fischeri than the identified cluster. The coding sequence of ftmPT3 was amplified by fusion PCR and overexpressed in Escherichia coli. The enzyme product of the soluble His(8)-FtmPT3 with verruculogen and dimethylallyl diphosphate (DMAPP) was identified unequivocally as fumitremorgin A by NMR and MS analyses. K(M) values of FtmPT3 were determined for verruculogen and DMAPP at 5.7 and 61.5 µM, respectively. Average turnover number (k(cat)) was calculated from kinetic parameters of verruculogen and DMAPP to be 0.069 s(-1). FtmPT3 also accepted biosynthetic precursors of fumitremorgin A, for example, fumitremorgin B and 12,13-dihydroxyfumitremorgin C, as substrates and catalyses their prenylation.
Subject(s)
Computational Biology , Dimethylallyltranstransferase/genetics , Dimethylallyltranstransferase/metabolism , Indoles/metabolism , Neosartorya/enzymology , Neosartorya/genetics , Chromosomes, Fungal/genetics , Culture Techniques , Dimethylallyltranstransferase/biosynthesis , Dimethylallyltranstransferase/chemistry , Indenes/metabolism , Kinetics , Multigene Family/genetics , Neosartorya/metabolism , Prenylation , Sequence Analysis , Substrate SpecificityABSTRACT
The prenyltransferase FtmPT1 from Aspergillus fumigatus is involved in the biosynthesis of fumitremorgin-type alkaloids and catalysed the regular C2-prenylation of brevianamide F (cyclo-L-Trp-L-Pro). It has been shown that FtmPT1 also accepted a number of other tryptophan-containing cyclic dipeptides and prenylated them, in the presence of dimethylallyl diphosphate, at C-2 of the indole nucleus. Detailed analysis of the incubation mixtures of FtmPT1 with these cyclic dipeptides revealed the presence of additional product peaks in the HPLC chromatograms. Seven regularly C3-prenylated hexahydropyrrolo[2,3-b]indoles were isolated and identified by HR-ESI-MS and NMR analyses including HMBC, HMQC and NOESY experiments. Further experiments proved that the C2- and C3-prenylated products are both independent enzyme products. To the best of our knowledge, this is the first report on the enzymatic formation of regularly C3-prenylated indolines. A reaction mechanism for both C2- and C3-prenylated derivatives was proposed.
Subject(s)
Aspergillus fumigatus/enzymology , Dimethylallyltranstransferase/chemistry , Hemiterpenes/chemistry , Indoles/chemistry , Organophosphorus Compounds/chemistry , Peptides, Cyclic/chemistry , Aspergillus fumigatus/chemistry , Chromatography, High Pressure Liquid , Dimethylallyltranstransferase/isolation & purification , Escherichia coli/genetics , Magnetic Resonance Spectroscopy , Prenylation , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Spectrometry, Mass, Electrospray Ionization , Stereoisomerism , Substrate SpecificityABSTRACT
Non-ribosomal peptide synthetases (NRPSs) are important enzymes of production machinery for natural products including clinically used antibiotics, antifungal, and anticancer agents. NRPS products are usually further modified by tailoring enzymes, resulting in the formation of diverse structures. We demonstrate here the production and isolation of metabolites produced by two bi-modular NRPSs, i.e., acetylaszonalenin and fumitremorgin-type alkaloids in Neosartorya fischeri NRRL181.
Subject(s)
Chemical Fractionation/methods , Neosartorya/metabolism , Peptide Synthases/metabolism , Chromatography, Gel , Chromatography, High Pressure Liquid , Culture Techniques , Indoles/analysis , Indoles/isolation & purification , Indoles/metabolism , Neosartorya/enzymology , Neosartorya/growth & development , Silicon Dioxide/chemistry , Spores, Fungal/growth & development , Spores, Fungal/metabolismABSTRACT
Three new sesquiterpenoids, named udasterpurenol A, udalactarane A, and udalactarane B, as well as the known compounds hyphodontal and sterpuric acid have been isolated from the basidiomycete Phlebia uda. These compounds represent the first natural products described from this species. The structures were elucidated by NMR spectroscopy and mass spectrometry. Udalactaranes A and B were isolated as mixtures with their respective epimeric acetals. These mixtures inhibited the spore germination of the plant pathogenic fungus Fusarium graminearum at 10 and 5 µg/mL, respectively, and were active against Jurkat cells with IC(50) values of 101 and 42 µM, respectively.
Subject(s)
Basidiomycota/chemistry , Sesquiterpenes/isolation & purification , Fusarium/drug effects , Humans , Inhibitory Concentration 50 , Jurkat Cells , Microbial Sensitivity Tests , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Sesquiterpenes/chemistry , Sesquiterpenes/pharmacologyABSTRACT
Fourteen tryptophan-containing cyclic dipeptides 1a-14a, including all four stereoisomers of cyclo-Trp-Pro and cyclo-Trp-Ala, were converted to their C2-regularly prenylated derivatives 1b-14b in the presence of dimethylallyl diphosphate by using the purified recombinant FtmPT1 as catalyst. The enzyme products were isolated on HPLC in preparative scales and their structures were elucidated by NMR and MS analyses. The cytotoxic effects of the prenylated products and their substrates were tested with human leukemia K562 and ovarian cancer A2780 sens and A2780 CisR cell lines. Preliminary results have been clearly shown that prenylation at C2 led to a significant increase of the cytotoxicity of the tested cyclic dipeptides in all the 14 cases. The second amino acid and the stereochemistry of tryptophan moiety of the cyclic dipeptides showed less influence on the cytotoxicity of the tested compounds.
Subject(s)
Dimethylallyltranstransferase/chemistry , Dipeptides/chemical synthesis , Fungal Proteins/chemistry , Peptides, Cyclic/chemical synthesis , Tryptophan/chemistry , Alanine/chemistry , Biocatalysis , Cell Line, Tumor , Cell Survival/drug effects , Chromatography, High Pressure Liquid , Dipeptides/pharmacology , Hemiterpenes/chemistry , Humans , Indoles/chemistry , Inhibitory Concentration 50 , Magnetic Resonance Spectroscopy , Organophosphorus Compounds/chemistry , Peptides, Cyclic/pharmacology , Prenylation , Proline/chemistry , Recombinant Proteins/chemistry , Stereoisomerism , Structure-Activity Relationship , Substrate SpecificityABSTRACT
From three basidiomycetes, Xeromphalina sp., Stereum sp., and Pleurocybella porrigens, six triquinane sesquiterpenes with unprecendented modifications and a rearranged sesquiterpene related to coriolin C have been isolated. Their isolation, structure elucidation, and biological evaluation are described.