Electrochemical and functional characterization of the proline dehydrogenase domain of the PutA flavoprotein from Escherichia coli.
Electrochemical and functional characterization of the proline dehydrogenase domain of the PutA flavoprotein from Escherichia coli.
复制标题
大肠杆菌 PutA 黄素蛋白脯氨酸脱氢酶结构域的电化学和功能表征。
DOI：
10.1021/bi025706f
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发表时间：
2002
期刊：
影响因子：
2.9
通讯作者：
Becker,DonaldF
中科院分区：
文献类型：
--
作者：
Vinod,MadhavanP;Bellur,Padmanetra;Becker,DonaldF
大肠杆菌（Escherichia coli）的多功能PutA黄素蛋白是一种具有脯氨酸脱氢酶（PDH）和Δ1-吡咯啉-5-羧酸脱氢酶（P5 CDH）活性的外周膜结合酶。除了其酶功能，PutA显示DNA结合活性，并通过与putregulon的控制区DNA（putintergenic DNA）结合来抑制脯氨酸催化剂。目前，关于PutA的结构-功能关系的信息来自一级结构分析。为了进一步了解PutA的功能组织，我们的目标是将PutA分解为不同的域并分别表征它们。在这里，我们报告了一种双功能脯氨酸脱氢酶（PutA 669）的特性，它含有PutA蛋白的1 - 669位残基。PutA 669纯化为二聚体，并且具有比PutA高4倍的PDH比活性。如预期的，PutA 669缺乏P5 CDH活性。在pH7.5时，电位滴定法测得PutA 669结合FAD的双电子还原Em（E−FAD/E−FADH-）为−0.091 V，比PutA结合FAD的Em负15 mV。在25 °C下，在6.5 - 9.0的pH范围内测量Emfor PutA 669结合的FAD的pH行为，并显示出0.03 V/pH单位斜率。对PutA 669的DNA和膜结合特性的分析表明，它以与PutA相似的结合亲和力特异性结合putintergenic对照DNA。相反，我们没有观察到PutA 669与膜囊泡的功能关联。我们的结论是，PutA 669具有FAD结合和DNA结合的性质相媲美的PutA，但缺乏一个膜结合结构域所必需的稳定协会与膜。
The multifunctional PutA flavoprotein fromEscherichia coliis a peripherally membrane-bound enzyme that has both proline dehydrogenase (PDH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) activities. In addition to its enzymatic functions, PutA displays DNA-binding activity and represses proline catabolism by binding to the control region DNA of theputregulon (putintergenic DNA). Presently, information on structure−function relationships for PutA is derived from primary structure analysis. To gain further insight into the functional organization of PutA, our objective is to dissect PutA into different domains and to characterize them separately. Here, we report the characterization of a bifunctional proline dehydrogenase (PutA669) that contains residues 1−669 of the PutA protein. PutA669purifies as a dimer and has a PDH specific activity that is 4-fold higher than that of PutA. As anticipated, PutA669lacks P5CDH activity. At pH 7.5, anEm(E−FAD/E−FADH-) of −0.091 V for the two-electron reduction of PutA669-bound FAD was determined by potentiometric titrations, which is 15 mV more negative than theEmfor PutA-bound FAD. The pH behavior of theEmfor PutA669-bound FAD was measured in the pH range 6.5−9.0 at 25 °C and exhibited a 0.03 V/pH unit slope. Analysis of the DNA and membrane-binding properties of PutA669shows that it binds specifically to theputintergenic control DNA with a binding affinity similar to that of PutA. In contrast, we did not observe functional association of PutA669with membrane vesicles. We conclude that PutA669has FAD-binding and DNA-binding properties comparable to those of PutA but lacks a membrane-binding domain necessary for stable association with the membrane.