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PutA DNA-binding transcriptional repressor

Synonyms: PutA
PutA is a flavoprotein with mutually exclusive functions as a transcriptional repressor and membrane-associated enzyme. The switch between the two activities is due to conformational changes triggered by the redox state of FAD. In the presence of proline, PutA is associated with the cytoplasmic membrane and acts a bifunctional enzyme catalyzing both reactions of the |FRAME: PROUT-PWY-I| pathway: the oxidation of proline by proline dehydrogenase and subsequent oxidation to glutamate by pyrroline-5-carboxylate (P5C) dehydrogenase. The kinetics of the coupled reaction is best described by substrate channeling. In the absence of proline, PutA is cytoplasmic and functions as a transcriptional repressor of the put regulon. The N-terminal 47 residues with a ribbon-helix-helix fold contain the dimerization domain and the specific DNA-binding activity of PutA [3, 4, 5]. The Lys9 residue is essential for recognition of put promoter DNA [6]. Crystal structures of this domain have been solved [3, 6]. In the absence of proline, PutA binds to operator sequences in the putA-putP intergenic region and represses transcription, most likely by keeping RNA polymerase from binding to the putA promoter [3].
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Transcription factor      
TF conformation(s):
Name Conformation Type TF-Effector Interaction Type Apo/Holo Conformation Evidence (Confirmed, Strong, Weak) References
PutA Functional   nd [1]
Evolutionary Family: PutA
Sensing class: External sensing using transported metabolites
Connectivity class: Local Regulator
Gene name: putA
  Genome position: 1074920-1078882
  Length: 3963 bp / 1320 aa
Operon name: putA
TU(s) encoding the TF:
Transcription unit        Promoter

Regulated gene(s) putA, putP
Multifun term(s) of regulated gene(s) MultiFun Term (List of genes associated to the multifun term)
amino acids (1)
electron donors (1)
Transcription related (1)
repressor (1)
Porters (Uni-, Sym- and Antiporters) (1)
Regulated operon(s) putA, putP
First gene in the operon(s) putA, putP
Simple and complex regulons BasR,MarA,PutA
Simple and complex regulatory phrases Regulatory phrase (List of promoters regulated by the phrase)

Transcription factor regulation    

Transcription factor binding sites (TFBSs) arrangements

  Functional conformation Function Promoter Sigma factor Central Rel-Pos Distance to first Gene Genes Sequence
LeftPos RightPos Evidence (Confirmed, Strong, Weak) References
  PutA repressor putAp Sigma70 -31.5 -74.5 putA
1078954 1078959 [BPP], [GEA], [SM] [1], [2], [3]
  PutA repressor putAp Sigma70 -21.5 -64.5 putA
1078944 1078949 [BPP], [GEA], [SM] [3]
  PutA repressor putAp Sigma70 14.5 -29.5 putA
1078909 1078914 [BPP], [GEA], [SM] [3]
  PutA repressor putPp1 Sigma70 -81.5 -218.5 putP
1079084 1079089 [BPP], [GEA], [SM] [3]
  PutA repressor putPp1 Sigma70 -54.5 -191.5 putP
1079111 1079116 [BPP], [GEA], [SM] [3]

Alignment and PSSM for PutA TFBSs    

Aligned TFBS of PutA   

Position weight matrix (PWM). PutA matrix-quality result   
A	0	0	0	5	5	0
C	0	0	5	0	0	5
G	0	5	0	0	0	0
T	5	0	0	0	0	0

;	consensus.strict             	TGCAAC
;	consensus.strict.rc          	GTTGCA
;	consensus.IUPAC              	TGCAAC
;	consensus.IUPAC.rc           	GTTGCA
;	consensus.regexp             	TGCAAC
;	consensus.regexp.rc          	GTTGCA

PWM logo   


Evolutionary conservation of regulatory elements    
     Note: Evolutionary conservation of regulatory interactions and promoters is limited to gammaproteobacteria.
TF-target gene evolutionary conservation
Promoter-target gene evolutionary conservation


 [BPP] Binding of purified proteins

 [GEA] Gene expression analysis

 [SM] Site mutation


 [1] Becker DF., Thomas EA., 2001, Redox properties of the PutA protein from Escherichia coli and the influence of the flavin redox state on PutA-DNA interactions., Biochemistry 40(15):4714-21

 [2] Brown ED., Wood JM., 1992, Redesigned purification yields a fully functional PutA protein dimer from Escherichia coli., J Biol Chem 267(18):13086-92

 [3] Zhou Y., Larson JD., Bottoms CA., Arturo EC., Henzl MT., Jenkins JL., Nix JC., Becker DF., Tanner JJ., 2008, Structural basis of the transcriptional regulation of the proline utilization regulon by multifunctional PutA., J Mol Biol 381(1):174-88

 [4] Gu D, Zhou Y, Kallhoff V, Baban B, Tanner JJ, Becker DF, 2004, Identification and characterization of the DNA-binding domain of the multifunctional PutA flavoenzyme., J Biol Chem, 2004 Jul 23

 [5] Singh RK, Larson JD, Zhu W, Rambo RP, Hura GL, Becker DF, Tanner JJ, 2011, Small-angle X-ray scattering studies of the oligomeric state and quaternary structure of the trifunctional proline utilization A (PutA) flavoprotein from Escherichia coli., J Biol Chem, 2011 Dec 16

 [6] Larson JD, Jenkins JL, Schuermann JP, Zhou Y, Becker DF, Tanner JJ, 2006, Crystal structures of the DNA-binding domain of Escherichia coli proline utilization A flavoprotein and analysis of the role of Lys9 in DNA recognition., Protein Sci, 2006 Nov

 [7] Zhang W, Zhou Y, Becker DF, 2004, Regulation of PutA-membrane associations by flavin adenine dinucleotide reduction., Biochemistry, 2004 Oct 19

 [8] Vinod MP, Bellur P, Becker DF, 2002, Electrochemical and functional characterization of the proline dehydrogenase domain of the PutA flavoprotein from Escherichia coli., Biochemistry, 2002 May 21

 [9] Abrahamson JL, Baker LG, Stephenson JT, Wood JM, 1983, Proline dehydrogenase from Escherichia coli K12. Properties of the membrane-associated enzyme., Eur J Biochem, 1983 Jul 15

 [10] Moxley MA, Tanner JJ, Becker DF, 2011, Steady-state kinetic mechanism of the proline:ubiquinone oxidoreductase activity of proline utilization A (PutA) from Escherichia coli., Arch Biochem Biophys, 2011 Dec 15

 [11] Moxley MA, Becker DF, 2012, Rapid reaction kinetics of proline dehydrogenase in the multifunctional proline utilization A protein., Biochemistry, 2012 Jan 10

 [12] Moxley MA, Zhang L, Christgen S, Tanner JJ, Becker DF, 2017, Identification of a Conserved Histidine As Being Critical for the Catalytic Mechanism and Functional Switching of the Multifunctional Proline Utilization A Protein., Biochemistry, 2017 Jun 20

 [13] Wood JM, 1987, Membrane association of proline dehydrogenase in Escherichia coli is redox dependent., Proc Natl Acad Sci U S A, 1987 Jan

 [14] Brown ED, Wood JM, 1993, Conformational change and membrane association of the PutA protein are coincident with reduction of its FAD cofactor by proline., J Biol Chem, 1993 Apr 25

 [15] Zhu W, Becker DF, 2003, Flavin redox state triggers conformational changes in the PutA protein from Escherichia coli., Biochemistry, 2003 May 13

 [16] Zhu W, Becker DF, 2005, Exploring the proline-dependent conformational change in the multifunctional PutA flavoprotein by tryptophan fluorescence spectroscopy., Biochemistry, 2005 Sep 20

 [17] Zhang W, Zhang M, Zhu W, Zhou Y, Wanduragala S, Rewinkel D, Tanner JJ, Becker DF, 2007, Redox-induced changes in flavin structure and roles of flavin N(5) and the ribityl 2'-OH group in regulating PutA--membrane binding., Biochemistry, 2007 Jan 16

 [18] Zhu W, Haile AM, Singh RK, Larson JD, Smithen D, Chan JY, Tanner JJ, Becker DF, 2013, Involvement of the ?3-?3 loop of the proline dehydrogenase domain in allosteric regulation of membrane association of proline utilization A., Biochemistry, 2013 Jul 2

 [19] 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, 2017 Nov 28

 [20] Lee YH, Nadaraia S, Gu D, Becker DF, Tanner JJ, 2003, Structure of the proline dehydrogenase domain of the multifunctional PutA flavoprotein., Nat Struct Biol, 2003 Feb

 [21] Zhang M, White TA, Schuermann JP, Baban BA, Becker DF, Tanner JJ, 2004, Structures of the Escherichia coli PutA proline dehydrogenase domain in complex with competitive inhibitors., Biochemistry, 2004 Oct 5

 [22] Ostrander EL, Larson JD, Schuermann JP, Tanner JJ, 2009, A conserved active site tyrosine residue of proline dehydrogenase helps enforce the preference for proline over hydroxyproline as the substrate., Biochemistry, 2009 Feb 10

 [23] Srivastava D, Zhu W, Johnson WH Jr, Whitman CP, Becker DF, Tanner JJ, 2010, The structure of the proline utilization a proline dehydrogenase domain inactivated by N-propargylglycine provides insight into conformational changes induced by substrate binding and flavin reduction., Biochemistry, 2010 Jan 26

 [24] Baban BA, Vinod MP, Tanner JJ, Becker DF, 2004, Probing a hydrogen bond pair and the FAD redox properties in the proline dehydrogenase domain of Escherichia coli PutA., Biochim Biophys Acta, 2004 Sep 1

 [25] Ling M, Allen SW, Wood JM, 1994, Sequence analysis identifies the proline dehydrogenase and delta 1-pyrroline-5-carboxylate dehydrogenase domains of the multifunctional Escherichia coli PutA protein., J Mol Biol, 1994 Nov 11

 [26] Moxley MA, Sanyal N, Krishnan N, Tanner JJ, Becker DF, 2014, Evidence for hysteretic substrate channeling in the proline dehydrogenase and ?1-pyrroline-5-carboxylate dehydrogenase coupled reaction of proline utilization A (PutA)., J Biol Chem, 2014 Feb 7

 [27] Deutch CE, Hasler JM, Houston RM, Sharma M, Stone VJ, 1989, Nonspecific inhibition of proline dehydrogenase synthesis in Escherichia coli during osmotic stress., Can J Microbiol, 1989 Aug

 [28] Zhang L, Alfano JR, Becker DF, 2015, Proline metabolism increases katG expression and oxidative stress resistance in Escherichia coli., J Bacteriol, 2015 Feb

 [29] Wood JM., Zadworny D., 1980, Amplification of the put genes and identification of the put gene products in Escherichia coli K12., Can J Biochem 58(10):787-96

 [30] Maloy S, Stewart V, 1993, Autogenous regulation of gene expression., J Bacteriol, 1993 Jan

 [31] Commichau FM, Stülke J, 2008, Trigger enzymes: bifunctional proteins active in metabolism and in controlling gene expression., Mol Microbiol, 2008 Feb

 [32] Zhou Y, Zhu W, Bellur PS, Rewinkel D, Becker DF, 2008, Direct linking of metabolism and gene expression in the proline utilization A protein from Escherichia coli., Amino Acids, 2008 Nov

 [33] Tanner JJ, 2008, Structural biology of proline catabolism., Amino Acids, 2008 Nov

 [34] Becker DF, Zhu W, Moxley MA, 2011, Flavin redox switching of protein functions., Antioxid Redox Signal, 2011 Mar 15

 [35] Singh RK, Tanner JJ, 2012, Unique structural features and sequence motifs of proline utilization A (PutA)., Front Biosci (Landmark Ed), 2012 Jan 1

 [36] Arentson BW, Sanyal N, Becker DF, 2012, Substrate channeling in proline metabolism., Front Biosci (Landmark Ed), 2012 Jan 1

 [37] Liu LK, Becker DF, Tanner JJ, 2017, Structure, function, and mechanism of proline utilization A (PutA)., Arch Biochem Biophys, 2017 Oct 15

 [38] Tanner JJ, 2019, Structural Biology of Proline Catabolic Enzymes., Antioxid Redox Signal, 2019 Feb 1