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首页> 外文期刊>Biochemistry >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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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

机译：脯氨酸利用N-炔丙基甘氨酸失活的脯氨酸脱氢酶结构域的结构提供了对底物结合和黄素还原诱导的构象变化的见解

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摘要

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 ε-amino of Lys329. Furthermore, inactivation locks PutA into a conformation that may mimic the proline-reduced, membrane-associated form. The 2.15? resolution structure of the inactivated PRODH domain suggests that the initial events involved in broadcasting the reduced flavin state to the distal membrane-binding domain include major reorganization of the flavin ribityl chain, severe (35°) butterfly bending of the isoalloxazine ring, and disruption of an 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 β1-αl loop to complete the active site.
机译：来自大肠杆菌的脯氨酸利用A（PutA）是一种黄素蛋白，具有互斥的作用，作为put regulon的转录阻遏物和与膜相关的酶，催化脯氨酸氧化为谷氨酸。先前的研究表明，脯氨酸在脯氨酸脱氢酶（PRODH）活性位点的结合以及随后FAD的降低触发了增强PutA膜亲和力的整体构象变化。这些事件导致PutA从其阻遏物切换到其酶的作用，但该信号从活性位点传播到远端膜结合域的机制在很大程度上尚不清楚。在此显示，N-炔丙基甘氨酸通过将黄素N（5）原子与Lys329的ε-氨基共价连接，不可逆地灭活PutA。此外，失活将PutA锁定为可模拟脯氨酸还原的膜相关形式的构象。 2.15？ PRODH结构域失活的高分辨率结构表明，将减少的黄素状态传播到远端膜结合域涉及的初始事件包括黄素核糖基链的重大重组，异四恶嗪环的严重（35°）蝶形弯曲和包含黄素N（5）原子，Arg431和Asp370的静电网络。该结构还提供有关与底物结合相关的构象变化的信息。该分析表明，在没有底物的情况下，活性位点是不完全组装的，脯氨酸的结合将螺旋8和β1-α1环中的保守残基聚集在一起以完成活性位点。

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