The biosynthesis of the alpha,beta-epoxyketone pharmacophore in natural product proteasome inhibitors
Final Report Abstract
Protease inhibitors are used in the clinics for the treatment of various e.g. myeloma, diabetes, hypertension and viral infections. Notably, the development of such drugs has greatly been inspired by the discovery of protease inhibitors from natural sources. These natural products often exhibit their inhibitory properties against proteases by covalent warhead-mediated binding to their target enzymes. A prominent example is epoxomicin, a proteasome inhibitor with a α’,β’-epoxyketone warhead which lead to the development of the anti-cancer drug Kyprolis®. Our project aimed to investigate the biosynthesis of the epoxyketone warhead in detail with the prospect to identify and characterize new proteasome inhibitors by heterologous expression of homologous pathways. We performed comparative gene cluster analysis and comprehensive gene deletion experiments in the biosynthetic pathways of two epoxyketone proteaome inhibitors: eponemycin and epoxomicin. Additionally we fed 13C-isotope enriched precursors to cultures of the producing bacteria. These investigations strongly indicated that a single enzyme with homology to acyl-CoA dehydrogenases transforms a polyketide synthase-derived oxoacid precursor to the epoxyketone. Thereby EpxF and EpnF would function as unique decarboxylase-dehydrogenase-oxygenase enzymes in the epoxomicin and eponemycin pathway, respectively. Using EpnF as a probe we identified a homologous pathway in Actinomadura atramentaria DSM43919 and expressed this gene cluster in a heterologous host. To our surprise the orphan gene cluster did not produce an epoxyketone proteasome inhibitor but instead metalloproteinase inhibitors containing a hydroxamic acid warhead: the matylstatins. By gene deletion experiments and isotope enriched precursor feeding we found that MatG, the EpnF homolog, is responsible for the astonishing structural diversity observed in these molecules. We postulate that MatG does not form an epoxyketone but a highly reactive vinylketone intermediate which can be attacked by various nucleophiles. Moreover, we showed that the carbon skeleton of the rare N-hydroxy-2-pentyl-succinamic acid moiety featured by the matlystatins and the prominent metalloproteinase inhibitor actinonin is assembled by an unprecedented variation of the ethylmalonyl-CoA pathway. In 2015 and 2016 several studies were published by highly reputed international research groups, with direct relation to our research topics. This particularly overlapped with our attempted biochemical investigations of EpnF and EpxF. We thus reorganized our approach and shifted our biochemical expertise to another class of proteasome inhibitors. Much like eponemycin and epoxomicin, the belactosins and the cystargolides comprise a short peptide backbone linked to an electrophilic moiety via an amide bond. However, in this case the warhead consists of a β-lactone ring. Identification and analysis of the corresponding gene clusters indicated that both compounds are assembled by rare single-enzyme amino acid ligases. Feeding experiments with isotope-labeled precursors and in vitro biochemistry showed that the formation of the b-lactone warhead is a unique reminiscent of leucine biosynthesis, and involves the action of isopropylmalate synthase homologues. In a follow-up project, we are now proposing to explore the biosynthesis of the N-hydroxy-alkyl-succinamic acid moiety and the 2-carboxy-3-alkyl βlactone moieties in more detail. Intriguingly, essential transformations in both cases mirror key reactions of the bacterial primary metabolism. The characterization of novel variations of such pathways and their enzymatic components is of great interest to the synthetic biology community as it facilitates the de novo design of complex biosynthetic systems in bacterial chassis.
Publications
- Epoxomicin and Eponemycin Biosynthesis Involves gem-Dimethylation and an AcylCoA Dehydrogenase-like Enzyme. ChemBioChem 17:792-8 (2016)
Zettler J, Zubeil F, Kulik A, Grond S, Kaysser L
(See online at https://doi.org/10.1002/cbic.201500567) - Biosynthesis of the β-lactone proteasome inhibitors belactosins and cystargolides. Angewandte Chemie Internationale Edition 56(23):6665-8 (2017)
Wolf F, Bauer JS, Bendel TM, Kulik A, Kalinowski J, Gross H, Kaysser L
(See online at https://doi.org/10.1002/anie.201612076) - Genom-basierte Suche nach Protease-Inhibitoren aus bakteriellen Quellen.” Biospektrum (2017), 23(1):41-45
Wolf F, Kaysser L
(See online at https://doi.org/10.1007/s12268-017-0765-9) - Warhead biosynthesis and the origin of structural diversity in hydroxamate metalloproteinase inhibitors. Nature Communications 8(1):1965 (2017)
Leipoldt F, Santos-Aberturas J, Stegmann DP, Wolf F, Kulik A, Lacret R, Popadić D, Keinhörster D, Kirchner N, Bekiesch P, Gross H, Truman AW, Kaysser L
(See online at https://doi.org/10.1038/s41467-017-01975-6) - Characterization of the actinonin biosynthetic gene cluster. ChemBioChem (2018)
Wolf F, Leipoldt F, Kulik A, Wibberg D, Kalinowski J, Kaysser L
(See online at https://doi.org/10.1002/cbic.201800116)
