Structural and Functional Analyses of a Spiro-Carbon-Forming, Highly Promiscuous Epoxidase from Fungal Natural Product Biosynthesis
Structural and Functional Analyses of a Spiro-Carbon-Forming, Highly Promiscuous Epoxidase from Fungal Natural Product Biosynthesis
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真菌天然产物生物合成中螺碳形成、高度混杂的环氧酶的结构和功能分析
DOI：
10.1021/acs.biochem.0c00896
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发表时间：
2020
期刊：
影响因子：
2.9
通讯作者：
Watanabe Kenji
中科院分区：
文献类型：
--
作者：
Matsushita Takuma;Kishimoto Shinji;Hara Kodai;Hashimoto Hiroshi;Watanabe Kenji
真菌非核糖体肽的生物合成通常涉及氧化还原酶，如含黄素单加氧酶（FMO），以将复杂性引入核心化学结构。一个这样的例子是由各种氧化酶催化的螺碳的形成。由于许多具有复杂化学结构的螺环碳化合物具有重要的生物活性，因此了解螺环碳化合物的生物合成机制具有重要意义。我们之前鉴定了FqzB，一种来自烟喹唑啉生物合成途径的FMO，负责烟喹唑啉F的环氧化，其与烟曲霉素生物合成途径发生交联，通过前体烟曲霉素C的环氧化形成螺曲前列腺素A。使FqzB更有趣的是其宽松的底物特异性，其中它可以接受一系列其他底物，包括tryprostatin A和B沿着其原始底物fumiquinazoline F。在这里，我们的特点FqzB晶体学和研究FqzB及其位点特异性突变体动力学，以了解这种混杂的环氧酶的作品。此外，诱变研究以及Fqz B晶体结构与其已知底物螺旋前列腺素A和B以及烟曲霉素C和烟喹唑啉F之间的计算对接实验，提供了对底物识别的潜在模式和该环氧酶所表现出的广泛底物耐受性的来源的深入了解。这项研究作为进一步表征和工程的氧化还原酶，这具有潜在的效用，作为一个有价值的催化剂，具有广泛的底物耐受性和化学酶和生物合成应用的碳框架中引入化学复杂性的能力的基础。
Biosynthesis of fungal nonribosomal peptides frequently involves redox enzymes such as flavin-containing monooxygenase (FMO) to introduce complexity into the core chemical structure. One such example is the formation of spiro-carbons catalyzed by various oxidases. Because many chemically complex spiro-carbon-bearing natural products exhibit useful biological activities, understanding the mechanism of spiro-carbon biosynthesis is of great interest. We previously identified FqzB, an FMO from the fumiquinazoline biosynthetic pathway responsible for epoxidation of fumiquinazoline F that crosstalks with the fumitremorgin biosynthetic pathway to form spirotryprostatin A via epoxidation of the precursor fumitremorgin C. What makes FqzB more interesting is its relaxed substrate specificity, where it can accept a range of other substrates, including tryprostatins A and B along with its original substrate fumiquinazoline F. Here, we characterized FqzB crystallographically and examined FqzB and its site-specific mutants kinetically to understand how this promiscuous epoxidase works. Furthermore, the mutagenesis studies as well as computational docking experiments between the FqzB crystal structure and its known substrates spirotryprostatin A and B, as well as fumitremorgin C and fumiquinazoline F, provided insight into potential modes of substrate recognition and the source of broad substrate tolerance exhibited by this epoxidase. This study serves as a foundation for further characterization and engineering of this redox enzyme, which has potential utility as a valuable catalyst with broad substrate tolerance and an ability to introduce chemical complexity into carbon frameworks for chemoenzymatic and biosynthetic applications.