Discovery of the Membrane Binding Domain in Trifunctional Proline Utilization A.
Discovery of the Membrane Binding Domain in Trifunctional Proline Utilization A.
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DOI：
10.1021/acs.biochem.7b01008
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发表时间：
2017-11-28
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：
Christgen SL;Zhu W;Sanyal N;Bibi B;Tanner JJ;Becker DF
Escherichia coli proline utilization A (EcPutA) is the archetype of trifunctional PutA flavoproteins, which function both as regulators of the proline utilization operon and bifunctional enzymes that catalyze the four-electron oxidation of proline to glutamate. EcPutA shifts from a self-regulating transcriptional repressor to a bifunctional enzyme in a process known as functional switching. The flavin redox state dictates the function of EcPutA. Upon proline oxidation, the flavin becomes reduced, triggering a conformational change that causes EcPutA to dissociate from the put regulon and bind to the cellular membrane. Major structure/function domains of EcPutA have been characterized, including the DNA-binding domain, proline dehydrogenase (PRODH) and L-glutamate-γ-semialdehyde dehydrogenase catalytic domains, and an aldehyde dehydrogenase superfamily fold domain. Still lacking is an understanding of the membrane-binding domain, which is essential for EcPutA catalytic turnover and functional switching. Here, we provide evidence for a conserved C-terminal motif (CCM) in EcPutA having a critical role in membrane binding. Deletion of the CCM or replacement of hydrophobic residues with negatively charged residues within the CCM impairs EcPutA functional and physical membrane association. Furthermore, cell-based transcription assays and limited proteolysis indicate that the CCM is essential for functional switching. Using fluorescence resonance energy transfer involving dansyl-labeled liposomes, residues in the α-domain are also implicated in membrane binding. Taken together, these experiments suggest that the CCM and α-domain converge to form a membrane-binding interface near the PRODH domain. The discovery of the membrane-binding region will assist efforts to define flavin redox signaling pathways responsible for EcPutA functional switching.
Escherichia coli proline utilization A (EcPutA) is the archetype of trifunctional PutA flavoproteins, which function both as regulators of the proline utilization operon and bifunctional enzymes that catalyze the four-electron oxidation of proline to glutamate. EcPutA shifts from a self-regulating transcriptional repressor to a bifunctional enzyme in a process known as functional switching. The flavin redox state dictates the function of EcPutA. Upon proline oxidation, the flavin becomes reduced, triggering a conformational change that causes EcPutA to dissociate from the put regulon and bind to the cellular membrane. Major structure/function domains of EcPutA have been characterized, including the DNA-binding domain, proline dehydrogenase (PRODH) and L-glutamate-γ-semialdehyde dehydrogenase catalytic domains, and an aldehyde dehydrogenase superfamily fold domain. Still lacking is an understanding of the membrane-binding domain, which is essential for EcPutA catalytic turnover and functional switching. Here, we provide evidence for a conserved C-terminal motif (CCM) in EcPutA having a critical role in membrane binding. Deletion of the CCM or replacement of hydrophobic residues with negatively charged residues within the CCM impairs EcPutA functional and physical membrane association. Furthermore, cell-based transcription assays and limited proteolysis indicate that the CCM is essential for functional switching. Using fluorescence resonance energy transfer involving dansyl-labeled liposomes, residues in the α-domain are also implicated in membrane binding. Taken together, these experiments suggest that the CCM and α-domain converge to form a membrane-binding interface near the PRODH domain. The discovery of the membrane-binding region will assist efforts to define flavin redox signaling pathways responsible for EcPutA functional switching.
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DOI：
10.1016/s1387-2656(05)11004-7
发表时间：
2005-01-01
期刊：
BIOTECHNOLOGY ANNUAL REVIEW, VOL 11
影响因子：
--
作者：
Berridge, MV;Herst, PM;Tan, AS
通讯作者：
Tan, AS
影响因子：
4
作者：
Arentson BW;Hayes EL;Zhu W;Singh H;Tanner JJ;Becker DF
通讯作者：
Becker DF
影响因子：
3.9
作者：
Liu LK;Becker DF;Tanner JJ
通讯作者：
Tanner JJ
影响因子：
2.9
作者：
Arentson, Benjamin W.;Luo, Min;Pemberton, Travis A.;Tanner, John J.;Becker, Donald F.
通讯作者：
Becker, Donald F.
影响因子：
8
作者：
Larson, John D.;Jenkins, Jermaine L.;Tanner, John J.
通讯作者：
Tanner, John J.