In vitro biosynthesis of molecular warheads from hydroxamate- and β-lactone-containing protease inhibitors

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Small-molecule protease inhibitors from natural sources have inspired the development of numerous pharmaceutical agents. In many cases, these natural products exhibit their inhibitory properties by warhead-mediated binding to the active site of target enzymes. In this research program we are planning to reconstitute the biosynthesis of the warhead moieties of two well-studied compound classes: the hydroxamate metalloproteinase inhibitors and the ß-lactone proteasome inhibitors. In a DFG-funded project we recently found that the carbon skeleton of the N-hydroxy-2-pentyl-succinamic acid warhead of actinonin and matlystatin is assembled by an unprecedented variation of the ethylmalonyl-CoA pathway. This involves the reductive carboxylation of octenoyl-CoA followed by epimerization and 1,2-rearrangement of the hexylmalonyl-CoA product. Moreover, we showed that the formation of 2-carboxy-3-alkyl ß-lactone warhead of belactosin and cystargolide is reminiscent of leucine biosynthesis featuring the aldol reaction of acetyl-CoA with an alpha-ketoacid and the stereo-specific isomerisation of the 2-alkylmalate product to 3-alkylmalate. Thus, both pathways represent unique adaptions of central biochemical processes to the secondary metabolism in bacteria with high potential for synthetic metabolic engineering. In the current project we want to individually reconstitute the enzymatic reactions in the biosynthesis of N-hydroxy-2-pentyl-succinamic acid in vitro and get a detailed understanding of the 1,2-rearrangement reaction of (2R)-hexylmalonyl-CoA by mechanistic studies and protein crystallography. We plan to evaluate key enzymatic reactions in the construction of the 2-carboxy-3-alkyl ß-lactone scaffold for their biotechnological capabilities. Moreover, we will investigate the formation of the ß-lactone ring of the cystargolides and belactosins by in vitro biochemistry with the prospect to identify the first bona fide β-lactone synthetase in natural product biosynthesis. The proposed research project will give us detailed mechanistic insights into two unique biosynthetic pathways in the bacterial secondary metabolism. This information will allow us to implement synthetic biology strategies for the design and production of tailor-made non-natural derivatives of these pharmaceutically interesting compound classes and facilitate the discovery of novel protease inhibitors by genome mining.