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 proline binding. In the presence of proline, PutA is associated with the cytoplasmic membrane and acts a bifunctional enzyme catalyzing both reactions of the proline degradation pathway: the oxidation of proline by proline dehydrogenase and subsequent oxidation to glutamate by pyrroline-5-carboxylate (P5C) dehydrogenase. 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 . 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 .
The proline dehydrogenase activity resides in the amino-terminal 669 amino acids of PutA; a truncated protein retains proline dehydrogenase and DNA-binding activity but lacks membrane association and 1-pyrroline-5-carboxylate dehydrogenase activity .
Proline dehydrogenase activity requires the presence of an electron acceptor; in vivo, it is thought that the reduced FADH2 transfers electrons to the quinone pool in the cytoplasmic membrane, and finally to oxygen via the respiratory chain . The proline:ubiquinone oxidoreductase reaction proceeds via a rapid equilibrium ping-pong mechanism with proline and ubiquinone binding at two distinct sites . Association of the enzyme with the membrane is dependent on reduction of the FAD cofactor, which induces a change in the conformation of the protein [10, 11, 12]. Both proline binding and FAD reduction contribute to the conformational change . The membrane-associated form of PutA does not bind put promoter DNA; thus, the enzymatic and DNA-binding activities of PutA are mutually exclusive .Read more >
Crystal structures of the amino-terminal proline dehydrogenase domain have been reported [15, 16, 17, 18, 19], and site-directed mutants in various active site residues have been characterized [16, 17, 18, 20]. These studies resulted in a detailed picture of the mechanism of the redox-dependent structural changes of the protein and identified Y540 as an important substrate specificity determinant. A solution structure of the entire protein using small-angle X-ray scattering (SAXS) show that PutA is a symmetric V-shaped dimer. Parts of the C-terminal domain may function as a lid that covers the internal substrate-channeling cavity .
The C-terminal domain shows similarity to aldehyde dehydrogenases, indicating that the true substrate for the second enzymatic activity may be γ-glutamic semialdehyde, which is thought to spontaneously equilibrate with P5C .
Expression of putA is reduced by prolonged exposure to osmotic stress .
PutA: proline utilization 
Reviews: [24, 25, 26]
|Sensing class:||External sensing using transported metabolites|
|Connectivity class:||Local Regulator|
|Length:||3963 bp / 1320 aa|
|TU(s) encoding the TF:||
|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)
Porters (Uni-, Sym- and Antiporters) (1)
|Regulated operon(s)||putA, putP|
|First gene in the operon(s)||putA, putP|
|Simple and complex regulons|
|Simple and complex regulatory phrases||
Regulatory phrase (List of promoters regulated by the phrase)
|Functional conformation||Function||Promoter||Sigma factor||Central Rel-Pos||Distance to first Gene||Genes||Sequence||LeftPos||RightPos||Evidence (Confirmed, Strong, Weak)||References|
|1078954||1078959||[BPP], [GEA], [SM]||, , |
|1078944||1078949||[BPP], [GEA], [SM]|||
|1078909||1078914||[BPP], [GEA], [SM]|||
|1079084||1079089||[BPP], [GEA], [SM]|||
|1079111||1079116||[BPP], [GEA], [SM]|||
|Alignment and PSSM for PutA TFBSs|
|Aligned TFBS of PutA|
|Position weight matrix (PWM).|
A 1 0 0 0 0 5 5 0 0 C 0 0 0 0 5 0 0 5 3 G 3 4 0 5 0 0 0 0 0 T 1 1 5 0 0 0 0 0 2
|Evolutionary conservation of regulatory elements|
 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
 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. 279(30):31171-6
 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. 286(50):43144-53
 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. 15(11):2630-41
 Vinod MP., Bellur P., Becker DF., 2002, Electrochemical and functional characterization of the proline dehydrogenase domain of the PutA flavoprotein from Escherichia coli., Biochemistry. 41(20):6525-32
 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. 516(2):113-20
 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. 43(39):12539-48
 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. 46(2):483-91
 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. 48(5):951-9
 Srivastava D., Zhu W., Johnson WH., 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. 49(3):560-9
 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. 1701(1-2):49-59
 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. 243(5):950-6
 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. 35(8):779-85