Regulation of flavin dehydrogenase compartmentalization :: requirements for PutA-membrane association in Salmonella typhimurium
Regulation of flavin dehydrogenase compartmentalization :: requirements for PutA-membrane association in Salmonella typhimurium
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DOI：
10.1016/s0005-2736(99)00104-2
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发表时间：
1999-09-21
影响因子：
3.4
通讯作者：
Maloy, S
中科院分区：
文献类型：
--
作者：
Surber, MW;Maloy, S
PUTA是一种多功能的外周膜蛋白，在鼠伤寒沙门氏菌和大肠杆菌中既是一种自体转录抑制因子，也是一种催化脯氨酸两步转化为谷氨酸的酶。为了了解Puta如何与膜结合，我们确定了FAD氧化还原和膜成分在Puta-膜结合中的作用。Put操纵子的解抑制和Puta的膜结合都需要减少紧密结合的FAD。FADH(2)改变了PUTA的构象，导致疏水性增加。以往的研究使用酶活性作为膜结合的指标，并得出结论：膜-膜相互作用需要从还原的FAD向膜传递电子。然而，直接物理分析表明，尽管电子传递对Pro脱氢酶活性是必需的，但PuTA与膜小泡结合的直接物理分析表明，它本身不是必需的。此外，PuTA能有效地与脂质体结合，表明Puta-膜结合不需要与其他膜蛋白相互作用。含有泛醌和细胞色素bo的脂质体可以有效地重组PUTA的酶活性，证实了脯氨酸脱氢酶可以直接将电子传递到醌库。这些结果表明，膜-膜结合严格地归因于FAD降低所引发的蛋白质-脂质相互作用。(C)1999 Elsevier Science B.V.保留所有权利。
PutA is a multifunctional, peripheral membrane protein which functions both as an autogenous transcriptional repressor and the enzyme which catalyzes the two-step conversion of proline to glutamate in Salmonella typhimurium and Escherichia coli. To understand how PutA associates with the membrane, we determined the role of FAD redox and membrane components in PutA-membrane association. Reduction of the tightly bound FAD is required for both derepression of the put operon and membrane association of PutA. FADH(2) alters the conformation of PutA, resulting in an increased hydrophobicity. Previous studies used enzymatic activity as an assay for membrane association and concluded that electron transfer from the reduced FAD in PutA to the membrane is required for the PutA-membrane interaction. However, direct physical assays of PutA association with membrane vesicles from quinone deficient mutants demonstrated that although electron transfer is essential for proline dehydrogenase activity, it is not required for PutA-membrane association per se. Furthermore, PutA efficiently associated with liposomes, indicating that PutA-membrane association does not require interactions with other membrane proteins. PutA enzymatic activity can be efficiently reconstituted with liposomes containing ubiquinone and cytochrome bo, confirming that proline dehydrogenase can pass electrons directly to the quinone pool. These results indicate that PutA-membrane association is due strictly to a protein-lipid interaction initiated by reduction of FAD. (C) 1999 Elsevier Science B.V. All rights reserved.