|Gene:||putA||Accession Numbers: EG10801 (EcoCyc), b1014, ECK1005|
Synonyms: putC, poaA
Subunit composition of
fused PutA DNA-binding transcriptional repressor / proline dehydrogenase / 1-pyrroline-5-carboxylate dehydrogenase = [PutA]2
fused PutA transcriptional repressor / proline dehydrogenase / 1-pyrroline-5-carboxylate dehydrogenase = 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 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 [Gu04, Zhou08, Singh11a]. The Lys9 residue is essential for recognition of put promoter DNA [Larson06]. Crystal structures of this domain have been solved [Larson06, Zhou08]. 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 [Zhou08].
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 [Vinod02]. 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 [Abrahamson83]. The proline:ubiquinone oxidoreductase reaction proceeds via a rapid equilibrium ping-pong mechanism with proline and ubiquinone binding at two distinct sites [Moxley11]. 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 [Wood87, Brown93, Zhu03]. Both proline binding and FAD reduction contribute to the conformational change [Zhu05]. The membrane-associated form of PutA does not bind put promoter DNA; thus, the enzymatic and DNA-binding activities of PutA are mutually exclusive [Zhang04d].
Crystal structures of the amino-terminal proline dehydrogenase domain have been reported [Lee03, Zhang04e, Zhang07b, Ostrander09, Srivastava10], and site-directed mutants in various active site residues have been characterized [Zhang04e, Baban04, Zhang07b, Ostrander09]. 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 [Singh11a].
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 [Ling94].
Expression of putA is reduced by prolonged exposure to osmotic stress [Deutch89].
PutA: "proline utilization" [Wood80]
Gene Citations: [Nakao87a]
Locations: inner membrane, cytosol
|Map Position: [1,074,143 <- 1,078,105] (23.15 centisomes)||Length: 3963 bp / 1320 aa|
Molecular Weight of Polypeptide: 143.82 kD (from nucleotide sequence), 137.0 kD (experimental) [Brown92 ]
Molecular Weight of Multimer: 293.0 kD (experimental) [Brown92]
Unification Links: ASAP:ABE-0003424 , CGSC:333 , DIP:DIP-10620N , EchoBASE:EB0794 , EcoGene:EG10801 , EcoliWiki:B1014 , Mint:MINT-1267731 , ModBase:P09546 , OU-Microarray:b1014 , PortEco:putA , PR:PRO_000023647 , Pride:P09546 , Protein Model Portal:P09546 , RefSeq:NP_415534 , RegulonDB:EG10801 , SMR:P09546 , String:511145.b1014 , UniProt:P09546
Relationship Links: InterPro:IN-FAMILY:IPR002872 , InterPro:IN-FAMILY:IPR005933 , InterPro:IN-FAMILY:IPR010985 , InterPro:IN-FAMILY:IPR015590 , InterPro:IN-FAMILY:IPR016160 , InterPro:IN-FAMILY:IPR016161 , InterPro:IN-FAMILY:IPR016162 , InterPro:IN-FAMILY:IPR016163 , InterPro:IN-FAMILY:IPR024082 , InterPro:IN-FAMILY:IPR024089 , InterPro:IN-FAMILY:IPR024090 , InterPro:IN-FAMILY:IPR025703 , PDB:Structure:1K87 , PDB:Structure:1TIW , PDB:Structure:1TJ0 , PDB:Structure:1TJ1 , PDB:Structure:1TJ2 , PDB:Structure:2AY0 , PDB:Structure:2FZM , PDB:Structure:2FZN , PDB:Structure:2GPE , PDB:Structure:2RBF , PDB:Structure:3E2Q , PDB:Structure:3E2R , PDB:Structure:3E2S , PDB:Structure:3ITG , PDB:Structure:4JNY , PDB:Structure:4JNZ , Pfam:IN-FAMILY:PF00171 , Pfam:IN-FAMILY:PF01619 , Pfam:IN-FAMILY:PF14850 , Prosite:IN-FAMILY:PS00070 , Prosite:IN-FAMILY:PS00687
In Paralogous Gene Group: 95 (14 members)
|Biological Process:||GO:0010133 - proline catabolic process to glutamate
[UniProtGOA12, GOA01a, Wood80]
GO:0045892 - negative regulation of transcription, DNA-templated [Zhou08]
GO:0006351 - transcription, DNA-templated [UniProtGOA11a]
GO:0006355 - regulation of transcription, DNA-templated [UniProtGOA11a, GOA01a]
GO:0006560 - proline metabolic process [UniProtGOA11a]
GO:0006561 - proline biosynthetic process [GOA01a]
GO:0008152 - metabolic process [UniProtGOA11a, GOA01a]
GO:0055114 - oxidation-reduction process [UniProtGOA11a, GOA01a]
|Molecular Function:||GO:0000986 - bacterial-type RNA polymerase core promoter proximal region sequence-specific DNA binding
GO:0001141 - bacterial-type RNA polymerase core promoter proximal region sequence-specific DNA binding transcription factor activity involved in negative regulation of transcription [Zhou08]
GO:0003677 - DNA binding [UniProtGOA11a, Brown92]
GO:0003842 - 1-pyrroline-5-carboxylate dehydrogenase activity [GOA01a, Brown92]
GO:0004657 - proline dehydrogenase activity [GOA01a, Moxley11, Ostrander09, Graham84, Brown92]
GO:0043565 - sequence-specific DNA binding [Gu04]
GO:0050660 - flavin adenine dinucleotide binding [Brown92]
GO:0003700 - sequence-specific DNA binding transcription factor activity [GOA01a]
GO:0003824 - catalytic activity [UniProtGOA11a]
GO:0016491 - oxidoreductase activity [UniProtGOA11a, GOA01a]
GO:0016620 - oxidoreductase activity, acting on the aldehyde or oxo group of donors, NAD or NADP as acceptor [GOA01a]
|Cellular Component:||GO:0009898 - cytoplasmic side of plasma membrane
GO:0005737 - cytoplasm
GO:0005829 - cytosol [Graham84]
GO:0005886 - plasma membrane [Graham84]
|MultiFun Terms:||information transfer → RNA related → Transcription related|
|metabolism → carbon utilization → amino acids|
|metabolism → energy production/transport → electron donors|
|regulation → type of regulation → transcriptional level → repressor|
DNA binding site length: 6 base-pairs
Consensus DNA Binding Sequence: GTTGCA
|Growth Medium||Growth?||T (°C)||O2||pH||Osm/L||Growth Observations|
|LB enriched||Yes||37||Aerobic||6.95||Yes [Gerdes03, Comment 1]|
|LB Lennox||Yes||37||Aerobic||7||Yes [Baba06, Comment 2]|
|M9 medium with 1% glycerol||Yes||37||Aerobic||7.2||0.35||Yes [Joyce06, Comment 3]|
|MOPS medium with 0.4% glucose||Yes||37||Aerobic||7.2||0.22||Yes [Baba06, Comment 2] |
Yes [Feist07, Comment 4]
Enzymatic reaction of: proline dehydrogenase
Synonyms: proline oxidase, L-proline:(acceptor) oxidoreductase, PDH
EC Number: 220.127.116.11
Hydroxy-L-proline is utilized at 3% of the rate of L-proline [Scarpulla78].
Inhibition by cyanide of the activity measured in association with membranes suggests that electrons are transferred from FADH2 to a cyanide-sensitive terminal oxidase [Abrahamson83].
The highest catalytic efficiency has been observed with menadione and CoQ2 as the electron acceptor. The Km for proline is lowest with membrane vesicles as electron acceptor, at 1.5 mM [Moxley11].
Enzymatic reaction of: 1-pyrroline-5-carboxylate dehydrogenase
Synonyms: δ-1-pyrroline-5-carboxylate dehydrogenase, P5C dehydrogenase, 1-pyrroline-5-carboxylate:NAD+ oxidoreductase, P5CDH
EC Number: 18.104.22.168
The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the Enzyme Commission system.
The reaction is irreversible in the direction shown.
In Pathways: proline degradation
|Protein-Segment||228 -> 574|
|Protein-Segment||653 -> 1119|
10/20/97 Gene b1014 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10801; confirmed by SwissProt match.
Abrahamson83: 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;134(1);77-82. PMID: 6305659
Baba06: Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006). "Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection." Mol Syst Biol 2;2006.0008. PMID: 16738554
Baban04: 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. PMID: 15450175
Becker01: 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. PMID: 11294639
Brown93: 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;268(12);8972-9. PMID: 8473341
Deutch89: 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. PMID: 2684374
Feist07: Feist AM, Henry CS, Reed JL, Krummenacker M, Joyce AR, Karp PD, Broadbelt LJ, Hatzimanikatis V, Palsson BO (2007). "A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information." Mol Syst Biol 3;121. PMID: 17593909
Gerdes03: Gerdes SY, Scholle MD, Campbell JW, Balazsi G, Ravasz E, Daugherty MD, Somera AL, Kyrpides NC, Anderson I, Gelfand MS, Bhattacharya A, Kapatral V, D'Souza M, Baev MV, Grechkin Y, Mseeh F, Fonstein MY, Overbeek R, Barabasi AL, Oltvai ZN, Osterman AL (2003). "Experimental determination and system level analysis of essential genes in Escherichia coli MG1655." J Bacteriol 185(19);5673-84. PMID: 13129938
Graham84: Graham SB, Stephenson JT, Wood JM (1984). "Proline dehydrogenase from Escherichia coli K12. Reconstitution of a functional membrane association." J Biol Chem 1984;259(4);2656-61. PMID: 6321477
Gu04: 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. PMID: 15155740
Joyce06: Joyce AR, Reed JL, White A, Edwards R, Osterman A, Baba T, Mori H, Lesely SA, Palsson BO, Agarwalla S (2006). "Experimental and computational assessment of conditionally essential genes in Escherichia coli." J Bacteriol 188(23);8259-71. PMID: 17012394
Larson06: 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. PMID: 17001030
Ling94: 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;243(5);950-6. PMID: 7966312
Moxley11: 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. PMID: 22040654
Ostrander09: 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. PMID: 19140736
Scarpulla78: Scarpulla RC, Soffer RL (1978). "Membrane-bound proline dehydrogenase from Escherichia coli. Solubilization, purification, and characterization." J Biol Chem 1978;253(17);5997-6001. PMID: 355248
Singh11a: 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. PMID: 22013066
Srivastava10: 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. PMID: 19994913
Vinod02: 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. PMID: 12009917
Zhang04e: 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. PMID: 15449943
Zhang07b: 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. PMID: 17209558
Zhou08: 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. PMID: 18586269
Zhou08a: 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 35(4);711-8. PMID: 18324349
Zhu02: Zhu W, Gincherman Y, Docherty P, Spilling CD, Becker DF (2002). "Effects of proline analog binding on the spectroscopic and redox properties of PutA." Arch Biochem Biophys 408(1);131-6. PMID: 12485611
Zhu05: Zhu W, Becker DF (2005). "Exploring the proline-dependent conformational change in the multifunctional PutA flavoprotein by tryptophan fluorescence spectroscopy." Biochemistry 44(37);12297-306. PMID: 16156643
Ogasawara12: Ogasawara H, Shinohara S, Yamamoto K, Ishihama A (2012). "Novel regulation targets of the metal-response BasS-BasR two-component system of Escherichia coli." Microbiology 158(Pt 6);1482-92. PMID: 22442305
Pomposiello01: Pomposiello PJ, Bennik MH, Demple B (2001). "Genome-wide transcriptional profiling of the Escherichia coli responses to superoxide stress and sodium salicylate." J Bacteriol 183(13);3890-902. PMID: 11395452
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