Department of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan.
Biochemistry. 2020 Dec 29;59(51):4787-4792. doi: 10.1021/acs.biochem.0c00896. Epub 2020 Dec 17.
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.
真菌非核糖体肽的生物合成通常涉及黄素单加氧酶（FMO）等氧化还原酶，以在核心化学结构中引入复杂性。例如，各种氧化酶催化的螺碳的形成就是一个例子。由于许多具有复杂化学结构的含螺碳天然产物具有有用的生物活性，因此了解螺碳生物合成的机制非常重要。我们之前鉴定了 FqzB，它是来自 fumiquinazoline 生物合成途径的 FMO，负责 fumiquinazoline F 的环氧化，与 fumitremorgin 生物合成途径相互作用，通过 fumitremorgin C 的环氧化形成 spirotryprostatin A。使 FqzB 更有趣的是其宽松的底物特异性，它可以接受一系列其他底物，包括 tryprostatins A 和 B 以及其原始底物 fumiquinazoline F。在这里，我们通过晶体学对 FqzB 进行了表征，并对 FqzB 及其定点突变体进行了动力学研究，以了解这种混杂的环氧化酶如何发挥作用。此外，突变研究以及 FqzB 晶体结构与其已知底物 spirotryprostatin A 和 B 以及 fumitremorgin C 和 fumiquinazoline F 之间的计算对接实验，深入了解了这种环氧化酶的潜在底物识别模式和广泛的底物耐受性的来源。这项研究为进一步表征和工程化这种氧化还原酶奠定了基础，该酶具有广泛的底物耐受性和在碳框架中引入化学复杂性的能力，可用于化学酶和生物合成应用。
