Structure and Function of the a-Hydroxylation Bimodule of the Mupirocin Polyketide Synthase.
Structure and Function of the a-Hydroxylation Bimodule of the Mupirocin Polyketide Synthase.
复制标题
莫匹罗星聚酮合酶的α-羟基化双模块的结构和功能。
DOI：
10.1002/ange.202312514
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Winter AJ
中科院分区：
文献类型：
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作者：
Winter AJ
莫匹罗星是一种临床上重要的抗生素，由荧光假单胞菌中的atrans‐AT I型聚酮合酶（PKS）产生。主要生物活性代谢产物假单胞菌酸A（PA−A）组装在四取代四氢吡喃（THP）核心上，其中包含6-羟基，拟通过酰基载体蛋白（ACP）结合聚酮化合物链的硫酯的α-羟基化引入。在此，我们描述了一种体外方法，该方法将纯化的酶组分、化学合成、同位素标记、质谱和NMR与体内研究相结合，从而首次表征莫匹罗星生物合成途径的α-羟基化双模块。这些研究揭示了MupA羟基化的精确时间、底物特异性和构成该α-羟基化双模块的酶组分的ACP依赖性。此外，使用纯化的酶，它表明，MmpA KS 0显示宽松的底物特异性，这表明精确的时空控制ofin transMupA招聘的背景下的PKS。最后，多个模块间MupA/ACP相互作用的检测表明，这些双模块可能整合MupA到他们的组装。
Mupirocin is a clinically important antibiotic produced by atrans‐AT Type I polyketide synthase (PKS) inPseudomonas fluorescens. The major bioactive metabolite, pseudomonic acid A (PA−A), is assembled on a tetrasubstituted tetrahydropyran (THP) core incorporating a 6‐hydroxy group proposed to be introduced by α‐hydroxylation of the thioester of the acyl carrier protein (ACP) bound polyketide chain. Herein, we describe an in vitro approach combining purified enzyme components, chemical synthesis, isotopic labelling, mass spectrometry and NMR in conjunction with in vivo studies leading to the first characterisation of the α‐hydroxylation bimodule of the mupirocin biosynthetic pathway. These studies reveal the precise timing of hydroxylation by MupA, substrate specificity and the ACP dependency of the enzyme components that comprise this α‐hydroxylation bimodule. Furthermore, using purified enzyme, it is shown that the MmpA KS0shows relaxed substrate specificity, suggesting precise spatiotemporal control ofin transMupA recruitment in the context of the PKS. Finally, the detection of multiple intermodular MupA/ACP interactions suggests these bimodules may integrate MupA into their assembly.