Discovery of the Membrane Binding Domain in Trifunctional Proline Utilization A

Shelbi L. Christgen, Weidong Zhu, Nikhilesh Sanyal, Bushra Bibi, John J. Tanner, Donald F Becker

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)6292-6303
Number of pages12
JournalBiochemistry
Volume56
Issue number47
DOIs
StatePublished - Nov 28 2017

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Proline
Escherichia coli
Membranes
Proline Oxidase
Glutamate-5-Semialdehyde Dehydrogenase
Oxidation-Reduction
Proteolysis
Flavoproteins
Regulon
Oxidation
Aldehyde Dehydrogenase
Fluorescence Resonance Energy Transfer
Enzymes
Transcription
Operon
Liposomes
Glutamic Acid
Assays
Catalytic Domain
Association reactions

ASJC Scopus subject areas

  • Biochemistry

Cite this

Discovery of the Membrane Binding Domain in Trifunctional Proline Utilization A. / Christgen, Shelbi L.; Zhu, Weidong; Sanyal, Nikhilesh; Bibi, Bushra; Tanner, John J.; Becker, Donald F.

In: Biochemistry, Vol. 56, No. 47, 28.11.2017, p. 6292-6303.

Research output: Contribution to journalArticle

Christgen, SL, Zhu, W, Sanyal, N, Bibi, B, Tanner, JJ & Becker, DF 2017, 'Discovery of the Membrane Binding Domain in Trifunctional Proline Utilization A', Biochemistry, vol. 56, no. 47, pp. 6292-6303. https://doi.org/10.1021/acs.biochem.7b01008
Christgen, Shelbi L. ; Zhu, Weidong ; Sanyal, Nikhilesh ; Bibi, Bushra ; Tanner, John J. ; Becker, Donald F. / Discovery of the Membrane Binding Domain in Trifunctional Proline Utilization A. In: Biochemistry. 2017 ; Vol. 56, No. 47. pp. 6292-6303.
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N2 - 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.

AB - 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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