Increasing structural diversity of natural products by enzymatic or nonenzymatic reactions

Secondary metabolites are generally low-molecule-mass compounds, also known as natural products. So far, millions of natural products with remarkably structural diversity have been found in nature. These include, but are not limited to, polyketides, nonribosomal peptides, alkaloids, and terpenoids....

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Main Author: Liao, Ge
Contributors: Li, Shu-Ming (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2020
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Summary:Secondary metabolites are generally low-molecule-mass compounds, also known as natural products. So far, millions of natural products with remarkably structural diversity have been found in nature. These include, but are not limited to, polyketides, nonribosomal peptides, alkaloids, and terpenoids. The structural divergence of natural products begins with the formation of basic skeletons by different backbone enzymes using fundamental building blocks derived from primary metabolism. Following modifications of the pre-matured scaffolds are catalyzed by tailoring enzymes such as oxidoreductases and transferases, thus completing the biosynthesis of end products with vast diversity and complexity. Enzymes from natural product biosynthetic pathways are versatile biocatalysts due to the merits of high efficiency and specificity. Harnessing biocatalytic potential of enzymes through chemoenzymatic synthesis has proven to be a useful tool for enriching chemical libraries. In addition to enzymatic catalysis, the occurrence of nonenzymatic events has also been found during the post-biosynthetic processing of natural products. These nonenzymatic reactions often occur with the involvement of reactive biosynthetic intermediates. Full exploitation of these intermediates for chemical synthesis can be used as an updated strategy to expand the chemical variety of natural products. In a cooperation project with Dr. Peter Mai, five prenyltransferases (PTs) of the dimethylallyltryptophan synthase (DMATS) family, i.e. FtmPT1, BrePT, CdpC2PT, CdpNPT, and CdpC3PT, were selected for protein engineering. These PTs catalyze a regular or reverse transfer of the dimethylallyl residue (C5) from dimethylallyl diphosphate (DMAPP) to the C-2 or C-3 position of indolyl residues in cyclic dipeptides (CDPs). To switch their prenyl donor specificity from DMAPP to geranyl diphosphate (GPP), protein sequence alignments of the five PTs with those of AtaPT and FgaPT2 led to the identification of the gatekeeping residues at Met364 in FtmPT1, Ile337 in BrePT, Thr351 in CdpC2PT, Met349 in CdpNPT, and Phe335 in CdpC3PT. Replacing the respective key amino acids by glycine resulted in the construction of FtmPT1_M364G, BrePT_I337G, CdpC2PT_T351G, CdpNPT_M349G, and CdpC3PT_F335G. These mutants showed clearly improved activity toward GPP but reduced activity toward DMAPP. As a result, 42 geranylated derivatives were obtained from the incubation mixtures of the generated mutants with 15 tested CDPs in the presence of GPP and their structures were elucidated by NMR and MS analyses. When using cyclo-L-Trp-L-Trp as the acceptor and GPP as the donor, the transfer of geranyl moieties to all seven possible positions of the indole nucleus can be achieved by the engineered enzymes. Prior to our study, only limited numbers of geranylated indole derivatives have been reported. This study significantly increased the structural diversity of geranylated products by structure-based protein engineering of available dimethylallyl transferases. In a cooperation study with Dr. Jie Fan, the biosynthesis of peniphenone and penilactones in Penicillium crustosum PRB-2 was elucidated, which revealed occurrence of both enzymatic and nonenzymatic reactions during their formation. The hybrid PKS-NRPS TraA from the terrestric acid pathway is involved in the formation of crustosic acid, which undergoes decarboxylation by the nonheme FeII/2-OG-dependent oxygenase TraH and subsequent reduction by the flavin-containing oxidoreductase TraD to afford terrestric acid. Both acids are precursors of the r-butyrolactones. The nonreducing PKS ClaF from the clavatol pathway is responsible for the formation of clavatol, which is then oxidized to hydroxyclavatol by the nonheme FeII/2-OG-dependent oxygenase ClaD. Alongside with spontaneous dehydration of hydroxyclavatol to the reactive intermediate ortho-quinone methide (QM), nonenzymatic 1,4-Michael additions were initiated by the nucleophilic attack from the r-butyrolactones to the ortho-QMs, leading to the sequential formation of peniphenone D and penilactone A as well as penilactones D and B. In addition to utilizing enzymes from the natural biosynthetic machinery, an alternative strategy was used for structural diversification by taking advantage of reactive intermediates from the biosynthetic pathway of natural products. As mentioned above, the ortho-QM involved in the formation of peniphenone and penilactones was proven to be highly reactive and capable of reacting with r-butyrolactones spontaneously under very mild conditions. As a following work, the reactivity of the ortho-QM derived from hydroxyclavatol was tested with 101 natural products or natural product-like compounds. These include flavonoids, hydroxynaphthalenes, coumarins, xanthones, anthraquinones, phloroglucinols, phenolic acids, indole derivatives, tyrosine analogues, and quinolines. LC-MS analysis revealed product formation in the incubation mixtures of 85 tested reactants. 32 clavatol-containing products were isolated from 23 selected incubations and identified by NMR and MS analyses. The cross-coupling between the tested nucleophiles and the ortho-QM from hydroxyclavatol occurs preferentially via C-C bonds at the ortho- or para-position of phenolic hydroxyl groups and the C-2 position of the indole ring. The obtained products were also tested for their biological activities. This study proved the ortho-QM as an excellent Michael acceptor for a variety of substances, suggesting the utilization of the reactive biosynthetic intermediate for accessing chemical diversity of natural products.
Physical Description:357 Pages