Li Hao, Forson Benedicta, Eckshtain-Levi Meital, Valentino Hannah, Martín Del Campo Julia S, Tanner John J, Sobrado Pablo
Department of Biochemistry, Virginia Tech, Blacksburg, Virginia 24061, United States.
Departments of Biochemistry and Chemistry, University of Missouri, Columbia, Missouri 65211, United States.
Biochemistry. 2021 Jan 12;60(1):31-40. doi: 10.1021/acs.biochem.0c00679. Epub 2020 Dec 22.
The flavin reductase (FRED) and isobutylamine -hydroxylase (IBAH) from 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 is higher than its affinity for FAD, consistent with its function as a flavin reductase. Conversely, IBAH binds FAD more tightly than FAD, 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 μM. 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.
来自[具体来源未提及]的黄素还原酶（FRED）和异丁胺羟化酶（IBAH）构成了一个双组分、黄素依赖性单加氧酶系统，该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，它为IBAH提供还原型黄素，然后IBAH催化异丁胺（IBA）羟基化为异丁羟胺（IBHA）。在这项工作中，我们使用了几种互补方法来研究FRED：IBAH系统中的FAD结合、稳态和快速反应动力学以及酶-酶相互作用。FRED对FAD的亲和力高于对FAD的亲和力，这与其作为黄素还原酶的功能一致。相反，IBAH对FAD的结合比对FAD更紧密，这与其作为单加氧酶的作用一致。作为FAD还原的电子源，FRED对NADPH的偏好性比对NADH强28倍。等温滴定量热法用于研究FRED和IBAH的缔合。在存在氧化型或还原型FAD的情况下，FRED和IBAH以7 - 8 μM的解离常数缔合。在没有FAD的情况下未观察到相互作用。这些结果与在这个双组分系统中形成蛋白质-蛋白质复合物以将还原型黄素从还原酶直接转移到单加氧酶一致。
