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

天然来源的小分子蛋白酶抑制剂启发了许多药物的发展。在许多情况下，这些天然产物通过弹头介导的结合到靶酶的活性部位来展示它们的抑制特性。在这项研究计划中，我们计划重建两类研究很好的化合物的弹头部分的生物合成：异羟甲酸酯金属蛋白酶抑制剂和内酯蛋白酶体抑制剂。在DFG资助的一个项目中，我们最近发现，由放线菌素和Matlystatin组成的N-羟基-2-戊基-琥珀酸弹头的碳骨架是通过乙基丙二酰辅酶A途径前所未有的变异组装而成的。这包括辛烯基-辅酶A的还原羧化，然后是己基丙二酰辅酶A产物的异构化和1，2-重排。此外，我们还发现2-羧基-3-烷基-内酯弹头的形成类似于亮氨酸的生物合成，其特征是乙酰辅酶A与α-酮酸的Aldol反应以及2-烷基苹果酸产物的立体特异性异构化为3-烷基苹果酸。因此，这两条途径都代表了中央生化过程对具有高合成代谢工程潜力的细菌的次生代谢的独特适应。在本项目中，我们希望在体外重建N-羟基-2-戊基-琥珀酸生物合成中的酶反应，并通过机理研究和蛋白质结晶学对(2R)-己基丙二酰辅酶A的1，2-重排反应有一个详细的了解。我们计划评估构建2-羧基-3-烷基-内酯支架的关键酶反应，以了解它们的生物技术能力。此外，我们还将通过体外生物化学研究半胱氨酸内酯和熊果苷的β-内酯环的形成，以期在天然产物的生物合成中鉴定第一个真正的β-内酯合成酶。拟议的研究项目将使我们从机制上详细了解细菌次级代谢中两条独特的生物合成途径。这些信息将使我们能够实施合成生物学策略，以设计和生产这些具有药用价值的化合物类的定制非天然衍生物，并促进通过基因组挖掘发现新型蛋白酶抑制剂。

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.