Biochemical Characterization of the Two-Component Flavin-Dependent Monooxygenase Involved in Valanimycin Biosynthesis
Biochemical Characterization of the Two-Component Flavin-Dependent Monooxygenase Involved in Valanimycin Biosynthesis
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DOI：
10.1021/acs.biochem.0c00679
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发表时间：
2021-01-12
期刊：
影响因子：
2.9
通讯作者：
Sobrado, Pablo
中科院分区：
文献类型：
--
作者：
Li, Hao;Forson, Benedicta;Sobrado, Pablo
来自绿色链霉菌的黄素还原酶（FRED）和异丁胺N-羟化酶（IBAH）构成一个双组分黄素依赖性单加氧酶系统，催化缬氨霉素生物合成的第一步。FRED是一种氧化还原酶，其将还原的黄素提供给IBAH，然后IBAH催化异丁胺（IBA）羟基化为异丁基羟胺（IBHA）。在这项工作中，我们使用了几种互补的方法来研究FAD结合，稳态和快速反应动力学，以及酶-酶在FRED：IBAH系统中的相互作用。FRED对FAD（ox）的亲和力高于其对FAD（red）的亲和力，这与其作为黄素还原酶的功能一致。相反，IBAH与FAD（红色）的结合比FAD（ox）更紧密，这与其作为单加氧酶的作用一致。FRED表现出对NADPH的强烈偏好（28倍）超过NADH作为FAD还原的电子源。用等温滴定量热法研究了FRED与IBAH的缔合作用。在FAD存在下，无论是氧化的还是还原的，FRED和IBAH缔合，解离常数为7-8 μ M。在不存在FAD的情况下未观察到相互作用。这些结果是一致的形成的蛋白质-蛋白质复合物的还原酶的单加氧酶在这个双组分系统中的直接转移的黄素。
The flavin reductase (FRED) and isobutylamine N-hydroxylase (IBAH) from Streptomyces viridifaciens constitute a two-component, flavin-dependent monooxygenase system that catalyzes the first step in valanimycin biosynthesis. FRED is an oxidoreductase that provides the reduced flavin to IBAH, which then catalyzes the hydroxylation of isobutylamine (IBA) to isobutylhydroxylamine (IBHA). In this work, we used several complementary methods to investigate FAD binding, steady-state and rapid reaction kinetics, and enzyme-enzyme interactions in the FRED:IBAH system. The affinity of FRED for FAD(ox) is higher than its affinity for FAD(red), consistent with its function as a flavin reductase. Conversely, IBAH binds FAD(red) more tightly than FAD(ox), consistent with its role as a monooxygenase. FRED exhibits a strong preference (28-fold) for NADPH over NADH as the electron source for FAD reduction. Isothermal titration calorimetry was used to study the association of FRED and IBAH. In the presence of FAD, either oxidized or reduced, FRED and IBAH associate with a dissociation constant of 7-8 mu M. No interaction was observed in the absence of FAD. These results are consistent with the formation of a protein-protein complex for direct transfer of reduced flavin from the reductase to the monooxygenase in this two-component system.