Moxley Michael A, Zhang Lu, Christgen Shelbi, Tanner John J, Becker Donald F
Department of Biochemistry, Redox Biology Center, University of Nebraska-Lincoln , Lincoln, Nebraska 68588, United States.
Department of Biochemistry, University of Missouri-Columbia , Columbia, Missouri 65211, United States.
Biochemistry. 2017 Jun 20;56(24):3078-3088. doi: 10.1021/acs.biochem.7b00046. Epub 2017 Jun 8.
Proline utilization A from Escherichia coli (EcPutA) is a multifunctional flavoenzyme that oxidizes proline to glutamate through proline dehydrogenase (PRODH) and Δ-pyrroline-5-carboxylate dehydrogenase (P5CDH) activities, while also switching roles as a DNA-bound transcriptional repressor and a membrane-bound catabolic enzyme. This phenomenon, termed functional switching, occurs through a redox-mediated mechanism in which flavin reduction triggers a conformational change that increases EcPutA membrane binding affinity. Structural studies have shown that reduction of the FAD cofactor causes the ribityl moiety to undergo a crankshaft motion, indicating that the orientation of the ribityl chain is a key element of PutA functional switching. Here, we test the role of a conserved histidine that bridges from the FAD pyrophosphate to the backbone amide of a conserved leucine residue in the PRODH active site. An EcPutA mutant (H487A) was characterized by steady-state and rapid-reaction kinetics, and cell-based reporter gene experiments. The catalytic activity of H487A is severely diminished (>50-fold) with membrane vesicles as the electron acceptor, and H487A exhibits impaired lipid binding and in vivo transcriptional repressor activity. Rapid-reaction kinetic experiments demonstrate that H487A is 3-fold slower than wild-type EcPutA in a conformational change step following reduction of the FAD cofactor. Furthermore, the reduction potential (E) of H487A is ∼40 mV more positive than that of wild-type EcPutA, and H487A has an attenuated ability to catalyze the reverse PRODH chemical step of reoxidation by P5C. In this process, significant red semiquinone forms in contrast to the same reaction with wild-type EcPutA, in which facile two-electron reoxidation occurs without the formation of a measurable amount of semiquinone. These results indicate that His487 is critically important for the proline/P5C chemical step, conformational change kinetics, and functional switching in EcPutA.
来自大肠杆菌的脯氨酸利用蛋白A（EcPutA）是一种多功能黄素酶，它通过脯氨酸脱氢酶（PRODH）和Δ-吡咯啉-5-羧酸脱氢酶（P5CDH）的活性将脯氨酸氧化为谷氨酸，同时还兼具作为与DNA结合的转录阻遏物和与膜结合的分解代谢酶的作用。这种现象被称为功能转换，它通过一种氧化还原介导的机制发生，其中黄素还原引发构象变化，从而增加EcPutA与膜的结合亲和力。结构研究表明，FAD辅因子的还原会导致核糖醇部分发生曲轴运动，这表明核糖醇链的取向是PutA功能转换的关键因素。在这里，我们测试了一个保守组氨酸的作用，该组氨酸从FAD焦磷酸桥接到PRODH活性位点中一个保守亮氨酸残基的主链酰胺上。通过稳态和快速反应动力学以及基于细胞的报告基因实验对EcPutA突变体（H487A）进行了表征。以膜囊泡作为电子受体时，H487A的催化活性严重降低（>50倍），并且H487A表现出脂质结合受损和体内转录阻遏活性受损。快速反应动力学实验表明，在FAD辅因子还原后的构象变化步骤中，H487A比野生型EcPutA慢3倍。此外，H487A的还原电位（E）比野生型EcPutA更正约40 mV，并且H487A催化P5C再氧化的反向PRODH化学步骤的能力减弱。在这个过程中，与野生型EcPutA的相同反应形成了显著的红色半醌，在野生型EcPutA的反应中，容易发生双电子再氧化而不形成可测量量的半醌。这些结果表明，His487对于EcPutA中的脯氨酸/P5C化学步骤、构象变化动力学和功能转换至关重要。
