Chemical Diversification Based on Substrate Promiscuity of a Standalone Adenylation Domain in a Reconstituted NRPS System.
Chemical Diversification Based on Substrate Promiscuity of a Standalone Adenylation Domain in a Reconstituted NRPS System.
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基于重构 NRPS 系统中独立腺苷酸化结构域的底物混杂性的化学多样化。
DOI：
10.1021/acschembio.8b00938
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发表时间：
2019-01
影响因子：
4
通讯作者：
Jing He
中科院分区：
文献类型：
--
作者：
Mengyi Zhu;Lijuan Wang;Jing He
硫解链霉菌中非核糖体肽合成酶（NRPS）装配线（sfa）指导二异腈查尔基SF 2768（1）的形成，其特征在于异源表达和定向基因敲除。本文中，异源表达菌株和原始宿主的差异代谢分析导致分离出具有N-异戊酰基而不是3-异氰基丁酰基侧链的SF 2768类似物（2，sfa的副产物）。Sfa的生物合成逻辑和1和2之间的结构差异表明腺苷酸形成酶SfaB的底物混杂性。SfaB和一个成功重建的NRPS系统，包括一个四酶级联的进一步底物范围调查，使不同的羧酸结构单元纳入肽支架，并因此产生30个非天然产物。这种基于腺苷酸化结构域的底物灵活性和体外重构的结构多样化策略可以应用于其他腺苷酸化引发途径，从而为多样性导向的全合成提供补充方法。此外，通过两个关键醛中间体（2a和2b）的衍生化验证了推定赖氨酸δ-羟化酶SfaE的生物催化过程，从而扩展了酶促C-H键活化的工具包。
A nonribosomal peptide synthetase (NRPS) assembly line ( sfa) in Streptomyces thioluteus that directs the formation of the diisonitrile chalkophore SF2768 (1) has been characterized by heterologous expression and directed gene knockouts. Herein, differential metabolic analysis of the heterologous expression strain and the original host led to the isolation of an SF2768 analogue (2, a byproduct of sfa) that possesses N-isovaleryl rather than 3-isocyanobutyryl side chains. The proposed biosynthetic logic of sfa and the structural difference between 1 and 2 suggested substrate promiscuity of the adenylate-forming enzyme SfaB. Further substrate scope investigation of SfaB and a successfully reconstituted NRPS system including a four-enzyme cascade enabled incorporation of diverse carboxylic acid building blocks into peptide scaffolds, and 30 unnatural products were thus generated. This structural diversification strategy based on substrate flexibility of the adenylation domain and in vitro reconstitution can be applied to other adenylation-priming pathways, thus providing a supplementary method for diversity-oriented total synthesis. Additionally, the biocatalytic process of the putative lysine δ-hydroxylase SfaE was validated through the derivatization of two key aldehyde intermediates (2a and 2b), thereby expanding the toolkit of enzymatic C-H bond activation.
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