Identification of a Conserved Histidine As Being Critical for the Catalytic Mechanism and Functional Switching of the Multifunctional Proline Utilization A Protein.
Identification of a Conserved Histidine As Being Critical for the Catalytic Mechanism and Functional Switching of the Multifunctional Proline Utilization A Protein.
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DOI：
10.1021/acs.biochem.7b00046
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发表时间：
2017-06-20
期刊：
影响因子：
2.9
通讯作者：
Becker DF
中科院分区：
文献类型：
--
作者：
Moxley MA;Zhang L;Christgen S;Tanner JJ;Becker DF
来自大肠杆菌（EcPutA）的脯氨酸利用A （Proline utilization A）是一种多功能黄酶，它通过脯氨酸脱氢酶（PRODH）和Δ1-pyrroline-5-carboxylate脱氢酶（P5CDH）活性将脯氨酸氧化为谷氨酸，同时还可以转换为DNA结合转录抑制因子和膜结合分解代谢酶。这种现象被称为功能转换，通过氧化还原介导的机制发生，其中黄素还原触发构象变化，增加EcPutA膜结合亲和力。结构研究表明，FAD辅因子的减少导致ribityl部分发生曲轴运动，这表明ribityl链的取向是PutA功能切换的关键因素。在这里，我们测试了一个保守的组氨酸的作用，它将FAD焦磷酸盐连接到PRODH活性位点上一个保守的亮氨酸残基的主酰胺上。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。以膜泡为电子受体，H487A的催化活性严重降低（bbb50倍），H487A表现出脂质结合和体内转录抑制活性受损。快速反应动力学实验表明，H487A在FAD辅因子减少后的构象变化步骤比野生型EcPutA慢3倍。此外，H487A的还原电位（Em）比野生型EcPutA高约40 mv，并且H487A催化PRODH逆向化学步骤（即P5C的再氧化）的能力减弱。在此过程中，与野生型EcPutA的相同反应相反，形成显著的红色半醌，在野生型EcPutA中，容易发生2电子再氧化而不形成可测量的半醌。这些结果表明，His487对于脯氨酸/P5C的化学步骤、构象变化动力学和EcPutA的功能转换至关重要。
Proline utilization A from Escherichia coli (EcPutA) is a multifunctional flavoenzyme that oxidizes proline to glutamate through proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) activities, while also switching roles as a DNA bound transcriptional repressor and a membrane bound catabolic enzyme. This phenomenon, referred to as 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 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 (Em) of H487A is about 40-mV more positive than wild-type EcPutA and H487A has an attenuated ability to catalyze the reverse PRODH chemical step, i.e. re-oxidation by P5C. In this process, significant red semiquinone forms in contrast to the same reaction with wild-type EcPutA, in which facile 2-electron re-oxidation occurs without forming measurable semiquinone. These results indicate that His487 is critically important for the proline/P5C chemical step, conformational change kinetics, and functional switching in EcPutA.
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