Fungal indole alkaloid biogenesis through evolution of a bifunctional reductase/Diels-Alderase
Fungal indole alkaloid biogenesis through evolution of a bifunctional reductase/Diels-Alderase
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DOI：
10.1038/s41557-019-0326-6
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发表时间：
2019-11-01
期刊：
影响因子：
21.8
通讯作者：
Williams, Robert M.
中科院分区：
文献类型：
--
作者：
Dan, Qingyun;Newmister, Sean A.;Williams, Robert M.
异戊烯化吲哚生物碱如钙调素抑制剂malbrancheamides和驱虫剂paraherquamides具有很大的结构多样性和药用价值。在这里，我们报告通过互补的方法完成的malbrancheamide生物合成途径的完整说明。这些包括仿生全合成以获得天然生物碱和外消旋形式的生物合成中间体，以及体外酶促重构以提供获得天然对映体（+）-马糠酰胺的途径。从MaIG非核糖体肽合成酶（NRPS）还原切割L-Pro-L-Trp二肽，然后进行反向异戊二烯化和NRPS后级联反应，最终进行分子内[4+2]杂-Diels-桤木（IMDA）环化，得到双环[2.2.2]二氮杂辛烷支架。光学纯的（+）-前malbrancheamide的酶促组装涉及一种意想不到的两性离子中间体，其中MaIC作为双功能NADPH依赖性还原酶/Diels-Alderase催化对映选择性环加成。底物和产物复合物的晶体结构以及定点诱变和分子动力学模拟证明了MaIC和PhqE（其从paraherquamide途径的同系物）如何催化非对映体和对映体选择性环化，在这类重要的次级代谢产物的构建中。
Prenylated indole alkaloids such as the calmodulin-inhibitory malbrancheamides and anthelmintic paraherquamides possess great structural diversity and pharmaceutical utility. Here, we report complete elucidation of the malbrancheamide biosynthetic pathway accomplished through complementary approaches. These include a biomimetic total synthesis to access the natural alkaloid and biosynthetic intermediates in racemic form and in vitro enzymatic reconstitution to provide access to the natural antipode (+)-malbrancheamide. Reductive cleavage of an L-Pro-L-Trp dipeptide from the MaIG non-ribosomal peptide synthetase (NRPS) followed by reverse prenylation and a cascade of post-NRPS reactions culminates in an intramolecular [4+2] hetero-Diels-Alder (IMDA) cyclization to furnish the bicyclo[2.2.2]diazaoctane scaffold. Enzymatic assembly of optically pure (+)-premalbrancheamide involves an unexpected zwitterionic intermediate where MaIC catalyses enantioselective cycloaddition as a bifunctional NADPH-dependent reductase/Diels-Alderase. The crystal structures of substrate and product complexes together with site-directed mutagenesis and molecular dynamics simulations demonstrate how MaIC and PhqE (its homologue from the paraherquamide pathway) catalyse diastereo- and enantioselective cyclization in the construction of this important class of secondary metabolites.