PutA protein, a membrane-associated flavin dehydrogenase, acts as a redox-dependent transcriptional regulator.

Spicer, P Ostrovsky de; Maloy, Stanley

doi:10.1073/pnas.90.9.4295

Cited by 121 publications

(110 citation statements)

References 19 publications

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“…Therefore, this flexible region may be involved in transmitting signals from the PRODH active site to the DNA-binding domain that transform PutA from a DNA-binding protein to a membrane-bound enzyme. Models for how PutA switches from a DNA-binding protein to a peripheral position on the membrane generally involve proline reduction of FAD and a conformational change that increases the overall hydrophobicity of PutA and PutA-membrane associations (8,10). Proline and FAD reduction only slightly affect the overall dissociation constant of the PutA-DNA complex (ϳ2-fold increase) (14,38).…”

Section: Discussionmentioning

confidence: 99%

“…PutP encodes a high affinity sodium-proline transporter (9). PutA represses the expression of the put genes, which are transcribed in opposite directions, by binding to the put intergenic DNA region (1,7,8). The transcriptional repression of the put genes is relieved in the presence of proline by the translocation of PutA to a peripheral position on the membrane, where proline is efficiently converted to glutamate.…”

mentioning

confidence: 99%

“…The transcriptional repression of the put genes is relieved in the presence of proline by the translocation of PutA to a peripheral position on the membrane, where proline is efficiently converted to glutamate. Reduction of the flavin causes a conformational change in PutA that is thought to enhance PutA membrane associations, thereby disrupting PutA-DNA binding (8,10,11).…”

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confidence: 99%

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Identification and Characterization of the DNA-binding Domain of the Multifunctional PutA Flavoenzyme

Zhou

Kallhoff

et al. 2004

Journal of Biological Chemistry

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The PutA flavoprotein from Escherichia coli is a transcriptional repressor and a bifunctional enzyme that regulates and catalyzes proline oxidation. PutA represses transcription of genes putA and putP by binding to the control DNA region of the put regulon. The objective of this study is to define and characterize the DNA binding domain of PutA. The DNA binding activity of PutA, a 1320 amino acid polypeptide, has been localized to N-terminal residues 1-261. After exploring a potential DNA-binding region and an N-terminal deletion mutant of PutA, residues 1-90 (PutA90) were determined to contain DNA binding activity and stabilize the dimeric structure of PutA. Cell-based transcriptional assays demonstrate that PutA90 functions as a transcriptional repressor in vivo. The dissociation constant of PutA90 with the put control DNA was estimated to be 110 nM, which is slightly higher than that of the PutA-DNA complex (K d ϳ 45 nM). Primary and secondary structure analysis of PutA90 suggested the presence of a ribbonhelix-helix DNA binding motif in residues 1-47. To test this prediction, we purified and characterized PutA47. PutA47 is shown to purify as an apparent dimer, to exhibit in vivo transcriptional activity, and to bind specifically to the put control DNA. In gel-mobility shift assays, PutA47 was observed to bind cooperatively to the put control DNA with an overall dissociation constant of 15 nM for the PutA47-DNA complex. Thus, Nterminal residues 1-47 are critical for DNA-binding and the dimeric structure of PutA. These results are consistent with the ribbon-helix-helix family of transcription factors.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Identification and Characterization of the DNA-binding Domain of the Multifunctional PutA Flavoenzyme

Zhou

Kallhoff

et al. 2004

Journal of Biological Chemistry

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“…PutA proteins typically contain 1000-1300 amino-acid residues, with the PRODH domain located in the N-terminal half of the polypeptide chain and the P5CDH domain located in the C-terminal half. In addition to their PRODH and P5CDH activities, some PutA proteins, such as Escherichia coli PutA, serve as autogenous repressors and therefore contain an N-terminal DNA-binding domain (Gu et al, 2004;Menzel & Roth, 1981c;Ostrovsky De Spicer & Maloy;Becker & Thomas, 2001;Wood, 1981).…”

Section: Introductionmentioning

confidence: 99%

“…Several functional aspects of PutA have been investigated, including enzymatic activity (Menzel & Roth, 1981a,b;Surber & Maloy, 1998;Zhu et al, 2002;Baban et al, 2004), redox-linked conformational changes (Brown & Wood, 1993;Zhu & Becker, 2003;Zhang, Zhou et al, 2004) and transcriptional repression (Menzel & Roth, 1981c;Muro-Pastor & Maloy, 1995;Muro-Pastor et al, 1997;Ostrovsky De Spicer & Maloy, 1993;Gu et al, 2004). In addition, the crystal structure of the PRODH domain of E. coli PutA has been determined (Nadaraia et al, 2001;Lee et al, 2003;Zhang, White et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

Cloning, purification and crystallization ofThermus thermophilusproline dehydrogenase

White

Tanner

2005

Acta Cryst Sect F

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Nature recycles l-proline by converting it to l-glutamate. This four-electron oxidation process is catalyzed by the two enzymes: proline dehydrogenase (PRODH) and Á 1 -pyrroline-5-carboxylate dehydrogenase. This note reports the cloning, purification and crystallization of Thermus thermophilus PRODH, which is the prototype of a newly discovered superfamily of bacterial monofunctional PRODHs. The results presented here include production of a monodisperse protein solution through use of the detergent n-octyl -dglucopyranoside and the growth of native crystals that diffracted to 2.3 Å resolution at Advanced Light Source beamline 4.2.2. The space group is P2 1 2 1 2 1 , with unit-cell parameters a = 82.2, b = 89.6, c = 94.3 Å . The asymmetric unit is predicted to contain two protein molecules and 46% solvent. Molecularreplacement trials using a fragment of the PRODH domain of the multifunctional Escherichia coli PutA protein as the search model (24% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of T. thermophilus PRODH will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative.

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Signal Transduction by Trigger Enzymes: Bifunctional Enzymes and Transporters Controlling Gene Expression

Commichau

Stülke

2009

Bacterial Signaling

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PutA protein, a membrane-associated flavin dehydrogenase, acts as a redox-dependent transcriptional regulator.

Abstract: The proline utilization (put) operon of Salmonella typhimurium is transcriptionally repressed by PutA

Cited by 121 publications

References 19 publications

Identification and Characterization of the DNA-binding Domain of the Multifunctional PutA Flavoenzyme

Identification and Characterization of the DNA-binding Domain of the Multifunctional PutA Flavoenzyme

Cloning, purification and crystallization ofThermus thermophilusproline dehydrogenase

Signal Transduction by Trigger Enzymes: Bifunctional Enzymes and Transporters Controlling Gene Expression

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