Regulation of PutA - Membrane associations by flavin adenine dinucleotide reduction

Weimin Zhang, Yuzhen Zhou, Donald F Becker

Research output: Contribution to journalArticle

40 Citations (Scopus)

Abstract

Proline utilization A (PutA) from Escherichia coli is a multifunctional flavoprotein that is both a transcriptional repressor of the proline utilization (put) genes and a membrane-associated enzyme which catalyzes the 4-electron oxidation of proline to glutamate. Previously, proline was shown to induce PutA - membrane binding and alter the intracellular location and function of PutA. To distinguish the roles of substrate binding and FAD reduction in the mechanism of how PutA changes from a DNA-binding protein to a membrane-bound enzyme, the kinetic parameters of PutA - membrane binding were measured under different conditions using model lipid bilayers and surface plasmon resonance (SPR). The effects of proline, FAD reduction, and proline analogues on PutA - membrane associations were determined. Oxidized PutA shows no binding to E. coli polar lipid vesicles. In contrast, proline and sodium dithionite induce tight binding of PutA to the lipid bilayer with indistinguishable kinetic parameters and an estimated dissociation constant (KD) of <0.01 nM (pH 7.4) for the reduced PutA - lipid complex. Proline analogues such as L-THFA and DL-P5C also stimulate PutA binding to E. coli polar lipid vesicles with KD values ranging from ∼3.6 to 34 nM (pH 7.4) for the PutA - lipid complex. The greater PutA - membrane binding affinity (>300-fold) generated by FAD reduction relative to the nonreducing ligands demonstrates that FAD reduction controls PutA - membrane associations. On the basis of SPR kinetic analysis with differently charged lipid bilayers, the driving force for PutA - membrane binding is primarily hydrophobic. In the SPR experiments membrane-bound PutA did not bind put control DNA, confirming that the membrane-binding and DNA-binding activities of PutA are mutually exclusive. A model for the regulation of PutA is described in which the overall translocation of PutA from the cytoplasm to the membrane is driven by FAD reduction and the subsequent energy difference (∼24 kJ/mol) between PutA - membrane and PutA - DNA binding.

Original languageEnglish (US)
Pages (from-to)13165-13174
Number of pages10
JournalBiochemistry
Volume43
Issue number41
DOIs
StatePublished - Oct 19 2004

Fingerprint

Flavin-Adenine Dinucleotide
Proline
Membranes
Lipid bilayers
Surface Plasmon Resonance
Lipid Bilayers
Surface plasmon resonance
Kinetic parameters
Escherichia coli
A-Form DNA

ASJC Scopus subject areas

  • Biochemistry

Cite this

Regulation of PutA - Membrane associations by flavin adenine dinucleotide reduction. / Zhang, Weimin; Zhou, Yuzhen; Becker, Donald F.

In: Biochemistry, Vol. 43, No. 41, 19.10.2004, p. 13165-13174.

Research output: Contribution to journalArticle

Zhang, Weimin ; Zhou, Yuzhen ; Becker, Donald F. / Regulation of PutA - Membrane associations by flavin adenine dinucleotide reduction. In: Biochemistry. 2004 ; Vol. 43, No. 41. pp. 13165-13174.
@article{55fb42a3777a47c0804bd228b72e8dd3,
title = "Regulation of PutA - Membrane associations by flavin adenine dinucleotide reduction",
abstract = "Proline utilization A (PutA) from Escherichia coli is a multifunctional flavoprotein that is both a transcriptional repressor of the proline utilization (put) genes and a membrane-associated enzyme which catalyzes the 4-electron oxidation of proline to glutamate. Previously, proline was shown to induce PutA - membrane binding and alter the intracellular location and function of PutA. To distinguish the roles of substrate binding and FAD reduction in the mechanism of how PutA changes from a DNA-binding protein to a membrane-bound enzyme, the kinetic parameters of PutA - membrane binding were measured under different conditions using model lipid bilayers and surface plasmon resonance (SPR). The effects of proline, FAD reduction, and proline analogues on PutA - membrane associations were determined. Oxidized PutA shows no binding to E. coli polar lipid vesicles. In contrast, proline and sodium dithionite induce tight binding of PutA to the lipid bilayer with indistinguishable kinetic parameters and an estimated dissociation constant (KD) of <0.01 nM (pH 7.4) for the reduced PutA - lipid complex. Proline analogues such as L-THFA and DL-P5C also stimulate PutA binding to E. coli polar lipid vesicles with KD values ranging from ∼3.6 to 34 nM (pH 7.4) for the PutA - lipid complex. The greater PutA - membrane binding affinity (>300-fold) generated by FAD reduction relative to the nonreducing ligands demonstrates that FAD reduction controls PutA - membrane associations. On the basis of SPR kinetic analysis with differently charged lipid bilayers, the driving force for PutA - membrane binding is primarily hydrophobic. In the SPR experiments membrane-bound PutA did not bind put control DNA, confirming that the membrane-binding and DNA-binding activities of PutA are mutually exclusive. A model for the regulation of PutA is described in which the overall translocation of PutA from the cytoplasm to the membrane is driven by FAD reduction and the subsequent energy difference (∼24 kJ/mol) between PutA - membrane and PutA - DNA binding.",
author = "Weimin Zhang and Yuzhen Zhou and Becker, {Donald F}",
year = "2004",
month = "10",
day = "19",
doi = "10.1021/bi048596g",
language = "English (US)",
volume = "43",
pages = "13165--13174",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "41",

}

TY - JOUR

T1 - Regulation of PutA - Membrane associations by flavin adenine dinucleotide reduction

AU - Zhang, Weimin

AU - Zhou, Yuzhen

AU - Becker, Donald F

PY - 2004/10/19

Y1 - 2004/10/19

N2 - Proline utilization A (PutA) from Escherichia coli is a multifunctional flavoprotein that is both a transcriptional repressor of the proline utilization (put) genes and a membrane-associated enzyme which catalyzes the 4-electron oxidation of proline to glutamate. Previously, proline was shown to induce PutA - membrane binding and alter the intracellular location and function of PutA. To distinguish the roles of substrate binding and FAD reduction in the mechanism of how PutA changes from a DNA-binding protein to a membrane-bound enzyme, the kinetic parameters of PutA - membrane binding were measured under different conditions using model lipid bilayers and surface plasmon resonance (SPR). The effects of proline, FAD reduction, and proline analogues on PutA - membrane associations were determined. Oxidized PutA shows no binding to E. coli polar lipid vesicles. In contrast, proline and sodium dithionite induce tight binding of PutA to the lipid bilayer with indistinguishable kinetic parameters and an estimated dissociation constant (KD) of <0.01 nM (pH 7.4) for the reduced PutA - lipid complex. Proline analogues such as L-THFA and DL-P5C also stimulate PutA binding to E. coli polar lipid vesicles with KD values ranging from ∼3.6 to 34 nM (pH 7.4) for the PutA - lipid complex. The greater PutA - membrane binding affinity (>300-fold) generated by FAD reduction relative to the nonreducing ligands demonstrates that FAD reduction controls PutA - membrane associations. On the basis of SPR kinetic analysis with differently charged lipid bilayers, the driving force for PutA - membrane binding is primarily hydrophobic. In the SPR experiments membrane-bound PutA did not bind put control DNA, confirming that the membrane-binding and DNA-binding activities of PutA are mutually exclusive. A model for the regulation of PutA is described in which the overall translocation of PutA from the cytoplasm to the membrane is driven by FAD reduction and the subsequent energy difference (∼24 kJ/mol) between PutA - membrane and PutA - DNA binding.

AB - Proline utilization A (PutA) from Escherichia coli is a multifunctional flavoprotein that is both a transcriptional repressor of the proline utilization (put) genes and a membrane-associated enzyme which catalyzes the 4-electron oxidation of proline to glutamate. Previously, proline was shown to induce PutA - membrane binding and alter the intracellular location and function of PutA. To distinguish the roles of substrate binding and FAD reduction in the mechanism of how PutA changes from a DNA-binding protein to a membrane-bound enzyme, the kinetic parameters of PutA - membrane binding were measured under different conditions using model lipid bilayers and surface plasmon resonance (SPR). The effects of proline, FAD reduction, and proline analogues on PutA - membrane associations were determined. Oxidized PutA shows no binding to E. coli polar lipid vesicles. In contrast, proline and sodium dithionite induce tight binding of PutA to the lipid bilayer with indistinguishable kinetic parameters and an estimated dissociation constant (KD) of <0.01 nM (pH 7.4) for the reduced PutA - lipid complex. Proline analogues such as L-THFA and DL-P5C also stimulate PutA binding to E. coli polar lipid vesicles with KD values ranging from ∼3.6 to 34 nM (pH 7.4) for the PutA - lipid complex. The greater PutA - membrane binding affinity (>300-fold) generated by FAD reduction relative to the nonreducing ligands demonstrates that FAD reduction controls PutA - membrane associations. On the basis of SPR kinetic analysis with differently charged lipid bilayers, the driving force for PutA - membrane binding is primarily hydrophobic. In the SPR experiments membrane-bound PutA did not bind put control DNA, confirming that the membrane-binding and DNA-binding activities of PutA are mutually exclusive. A model for the regulation of PutA is described in which the overall translocation of PutA from the cytoplasm to the membrane is driven by FAD reduction and the subsequent energy difference (∼24 kJ/mol) between PutA - membrane and PutA - DNA binding.

UR - http://www.scopus.com/inward/record.url?scp=5444257841&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=5444257841&partnerID=8YFLogxK

U2 - 10.1021/bi048596g

DO - 10.1021/bi048596g

M3 - Article

VL - 43

SP - 13165

EP - 13174

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 41

ER -