The Structure of the Proline Utilization A Proline Dehydrogenase Domain Inactivated by N-Propargylglycine Provides Insight into Conformational Changes Induced by Substrate Binding and Flavin Reduction
The Structure of the Proline Utilization A Proline Dehydrogenase Domain Inactivated by N-Propargylglycine Provides Insight into Conformational Changes Induced by Substrate Binding and Flavin Reduction
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DOI：
10.1021/bi901717s
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发表时间：
2010-01-26
期刊：
影响因子：
2.9
通讯作者：
Tanner, John J.
中科院分区：
文献类型：
--
作者：
Srivastava, Dhiraj;Zhu, Weidong;Tanner, John J.
脯氨酸利用A（Proline utilization A，PutA）是大肠杆菌中的一种黄素蛋白，它既是一种调节子的转录抑制因子，又是一种膜相关酶，催化脯氨酸氧化为谷氨酸。先前的研究已经表明，脯氨酸脱氢酶（PRODH）活性位点中的脯氨酸的结合和随后的FAD的减少触发增强PutA-膜亲和力的全局构象变化。这些事件导致PutA从其阻遏物转变为其酶促作用，但这种信号从活性位点传播到远端膜结合结构域的机制在很大程度上是未知的。在此，显示N-炔丙基甘氨酸通过将黄素N（5）原子共价连接至Lys 329的ε-氨基而不可逆地使PutA失活。此外，失活锁定PutA的构象，可以模拟脯氨酸还原。膜结合型。2.15埃分辨率失活PRODH结构域的结构表明，最初的事件涉及将还原的黄素状态不良传播到远端膜结合结构域，包括黄素核糖基链的重大重组、异咯嗪环的严重（35度）蝴蝶弯曲，以及涉及黄素N（5）原子、Arg 431和Asp 370的所有静电网络的破坏。该结构还提供与底物结合相关的构象变化信息。该分析表明，活性位点在没有底物的情况下不完全组装，并且脯氨酸的结合将螺旋8和β 1-α 1环中的保守残基聚集在一起以完成活性位点。
Proline utilization A (PutA) from Escherichia coli is a flavoprotein that has mutually exclusive roles as a transcriptional repressor of the put regulon and a membrane-associated enzyme that catalyzes the oxidation of proline to glutamate. Previous studies have shown that the binding of proline in the proline dehydrogenase (PRODH) active site and subsequent reduction of the FAD trigger global conformational changes that enhance PutA-membrane affinity. These events cause PutA to switch from its repressor to its enzymatic role, but the mechanism by which this signal is propagated from the active site to the distal membrane-binding domain is largely unknown. Here, it is shown that N-propargylglycine irreversibly inactivates PutA by covalently linking the flavin N(5) atom to the epsilon-amino of Lys329. Furthermore, inactivation locks PutA into a conformation that may mimic the proline-reduced. membrane-associated form. The 2.15 angstrom resolution Structure Of the Inactivated PRODH domain Suggests that the initial events involved ill broadcasting the reduced flavin state to the distal membrane-binding domain Include major reorganization of the flavin ribityl chain, severe (35 degrees) butterfly bending of the isoalloxazine ring, and disruption of all electrostatic network involving the flavin N(5) atom, Arg431, and Asp370. The structure also provides information about conformational changes associated with substrate binding. This analysis suggests that the active site is incompletely assembled in the absence of the substrate, and the binding of proline draws together conserved residues In helix 8 and the beta 1-alpha 1 loop to complete the active site.