|Gene:||wrbA||Accession Numbers: EG11540 (EcoCyc), b1004, ECK0995|
Subunit composition of
NAD(P)H:quinone oxidoreductase = [WrbA]4
NAD(P)H:quinone oxidoreductase = WrbA
The purified WrbA protein has NAD(P)H:quinone oxidoreductase activity [Patridge06]. WrbA is related to the flavodoxin family of proteins [Grandori94]. Unlike the flavodoxins, WrbA does not have a stabilized semiquinone state. It rapidly takes up two electrons, generating the fully reduced form [Noll06]. Purified WrbA protein binds one FMN per monomer with a binding constant of 2 µM at room temperature, which is weaker than that of typical flavodoxins [Grandori98, Patridge06]. Binding of FMN appears to be pH-dependent [Patridge06], and it increases the thermal stability and promotes tetramerization of WrbA [Natalello07].
WrbA is a multimer in solution, existing in an equilibrium between the dimeric and tetrameric form [Grandori98, Patridge06]. Crystal structures show WrbA to be a dimer of dimers. Structural comparisons to flavodoxin and the mammalian NAD(P)H:quinone oxidoreductase Nqo1 allow interpretation of the differences in the cofactor requirements and the catalytic functions of these proteins [Wolfova07, Carey07]. Additional crystal structures of WrbA in complex with benzoquinone or NADH suggest that binding of quinones and NADH to the FMN cofactor is mutually exclusive [Andrade07]. Steady-state kinetic analysis suggests a ping-pong reaction mechanism and show two-plateau Michaelis-Menten plots that are dependent on the temperature at which the enzyme had been held. This result implies allosteric regulation of the enzyme [Kishko12].
Comparison of the crystal structures of the apo- and holoenzyme forms of WrbA led to improved understanding of the functional similarities and differences of WrbA compared to the flavodoxins [Wolfova09].
It was initially reported that WrbA copurifies with the Trp repressor protein TrpR and enhances the formation or stability of TrpR binding to its operator target sites [Yang93]. However, a later report showed that WrbA does not specifically affect the DNA binding affinity or mode of binding of TrpR [Grandori98]. WrbA alone does not bind to the trp operator DNA [Yang93]. The association between WrbA and TrpR observed by [Yang93] may therefore be due to structural rather than functional reasons [Grandori98].
Expression of wrbA is increased during stationary phase and is RpoS-dependent [Yang93, Lacour04] and Crl-dependent [Lelong07]. In a strain lacking the ClpP serine protease, the level of WrbA protein is decreased during exponential growth and late stationary phase [Weichart03]. Like other members of the RpoS regulon, the steady-state level of WrbA is increased by growth on acetate [Kirkpatrick01]. wrbA was predicted to be a target of the small RNA OxyS, and overexpression of OxyS decreases the expression of wrbA [Tjaden06]. Expression of wrbA is also likely directly repressed by ArcA-P [Liu04].
A wrbA null mutant has no growth defect when assayed with the Biolog system. Growth of the mutant strain is inhibited by N-trichloromethyl-mercapto-4-cyclohexene-1,2-dicarboximide and 8-hydroxyquinoline [Patridge06].
WrbA: "tryptophan (W) repressor-binding protein" [Yang93]
Locations: cytosol, membrane
|Map Position: [1,066,335 <- 1,066,931] (22.98 centisomes)||Length: 597 bp / 198 aa|
Molecular Weight of Polypeptide: 20.846 kD (from nucleotide sequence), 21 kD (experimental) [Yang93 ]
Unification Links: ASAP:ABE-0003392 , CGSC:31836 , DIP:DIP-36231N , EchoBASE:EB1502 , EcoGene:EG11540 , EcoliWiki:b1004 , Mint:MINT-1272067 , ModBase:P0A8G6 , OU-Microarray:b1004 , PortEco:wrbA , PR:PRO_000024221 , Pride:P0A8G6 , Protein Model Portal:P0A8G6 , RefSeq:NP_415524 , RegulonDB:EG11540 , SMR:P0A8G6 , String:511145.b1004 , UniProt:P0A8G6
Relationship Links: InterPro:IN-FAMILY:IPR008254 , InterPro:IN-FAMILY:IPR010089 , PDB:Structure:2R96 , PDB:Structure:2R97 , PDB:Structure:2RG1 , PDB:Structure:3B6I , PDB:Structure:3B6J , PDB:Structure:3B6K , PDB:Structure:3B6M , PDB:Structure:3ZHO , Pfam:IN-FAMILY:PF00258 , Prosite:IN-FAMILY:PS50902
Instance reaction of [a quinone + NAD(P)H + H+ → a quinol + NAD(P)+] (18.104.22.168):
|Biological Process:||GO:0006979 - response to oxidative stress
GO:0045892 - negative regulation of transcription, DNA-templated [GOA01]
GO:0055114 - oxidation-reduction process [UniProtGOA11a]
|Molecular Function:||GO:0003955 - NAD(P)H dehydrogenase (quinone) activity
[GOA06, GOA01a, Patridge06]
GO:0010181 - FMN binding [GOA01, Patridge06]
GO:0042802 - identical protein binding [Lasserre06]
GO:0000166 - nucleotide binding [UniProtGOA11a]
GO:0016491 - oxidoreductase activity [UniProtGOA11a, GOA01]
GO:0050660 - flavin adenine dinucleotide binding [GOA06]
GO:0050661 - NADP binding [GOA06]
GO:0051287 - NAD binding [GOA06]
|Cellular Component:||GO:0005829 - cytosol
[Ishihama08, LopezCampistrou05, Patridge06]
GO:0016020 - membrane [Lasserre06]
GO:0005737 - cytoplasm
|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]|
Enzymatic reaction of: NAD(P)H:quinone oxidoreductase
EC Number: 22.214.171.124
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 physiologically favored in the direction shown.
NADH is the preferred electron donor. Km values were measured for NADH and p-benzoquinone [Patridge06].
The midpoint potential at pH 7 is -115 mV [Zafar09].
pH(opt): 6-8 [Patridge06]
|Feature Class||Location||Attached Group||Citations||Comment|
|Chain||2 -> 198|
|Conserved-Region||4 -> 189|
|Nucleotide-Phosphate-Binding-Region||9 -> 14||FMN|
|Nucleotide-Phosphate-Binding-Region||77 -> 80||FMN|
|Nucleotide-Phosphate-Binding-Region||112 -> 118||FMN|
10/20/97 Gene b1004 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG11540; confirmed by SwissProt match.
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
Carey07: Carey J, Brynda J, Wolfova J, Grandori R, Gustavsson T, Ettrich R, Smatanova IK (2007). "WrbA bridges bacterial flavodoxins and eukaryotic NAD(P)H:quinone oxidoreductases." Protein Sci 16(10);2301-5. PMID: 17893367
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
Grandori98: Grandori R, Khalifah P, Boice JA, Fairman R, Giovanielli K, Carey J (1998). "Biochemical characterization of WrbA, founding member of a new family of multimeric flavodoxin-like proteins." J Biol Chem 273(33);20960-6. PMID: 9694845
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
Kirkpatrick01: Kirkpatrick C, Maurer LM, Oyelakin NE, Yoncheva YN, Maurer R, Slonczewski JL (2001). "Acetate and formate stress: opposite responses in the proteome of Escherichia coli." J Bacteriol 183(21);6466-77. PMID: 11591692
Kishko12: Kishko I, Harish B, Zayats V, Reha D, Tenner B, Beri D, Gustavsson T, Ettrich R, Carey J (2012). "Biphasic Kinetic Behavior of E. coli WrbA, an FMN-Dependent NAD(P)H:Quinone Oxidoreductase." PLoS One 7(8);e43902. PMID: 22952804
Lacour04: Lacour S, Landini P (2004). "SigmaS-dependent gene expression at the onset of stationary phase in Escherichia coli: function of sigmaS-dependent genes and identification of their promoter sequences." J Bacteriol 186(21);7186-95. PMID: 15489429
Lasserre06: Lasserre JP, Beyne E, Pyndiah S, Lapaillerie D, Claverol S, Bonneu M (2006). "A complexomic study of Escherichia coli using two-dimensional blue native/SDS polyacrylamide gel electrophoresis." Electrophoresis 27(16);3306-21. PMID: 16858726
Liu04: Liu X, De Wulf P (2004). "Probing the ArcA-P modulon of Escherichia coli by whole genome transcriptional analysis and sequence recognition profiling." J Biol Chem 279(13);12588-97. PMID: 14711822
LopezCampistrou05: Lopez-Campistrous A, Semchuk P, Burke L, Palmer-Stone T, Brokx SJ, Broderick G, Bottorff D, Bolch S, Weiner JH, Ellison MJ (2005). "Localization, annotation, and comparison of the Escherichia coli K-12 proteome under two states of growth." Mol Cell Proteomics 4(8);1205-9. PMID: 15911532
Natalello07: Natalello A, Doglia SM, Carey J, Grandori R (2007). "Role of flavin mononucleotide in the thermostability and oligomerization of Escherichia coli stress-defense protein WrbA." Biochemistry 46(2);543-53. PMID: 17209564
Noll06: Noll G, Kozma E, Grandori R, Carey J, Schodl T, Hauska G, Daub J (2006). "Spectroelectrochemical investigation of a flavoprotein with a flavin-modified gold electrode." Langmuir 22(5);2378-83. PMID: 16489832
Weichart03: Weichart D, Querfurth N, Dreger M, Hengge-Aronis R (2003). "Global role for ClpP-containing proteases in stationary-phase adaptation of Escherichia coli." J Bacteriol 185(1);115-25. PMID: 12486047
Wolfova07: Wolfova J, Mesters JR, Brynda J, Grandori R, Natalello A, Carey J, Kuta Smatanova I (2007). "Crystallization and preliminary diffraction analysis of Escherichia coli WrbA in complex with its cofactor flavin mononucleotide." Acta Crystallogr Sect F Struct Biol Cryst Commun 63(Pt 7);571-5. PMID: 17620713
Wolfova09: Wolfova J, Smatanova IK, Brynda J, Mesters JR, Lapkouski M, Kuty M, Natalello A, Chatterjee N, Chern SY, Ebbel E, Ricci A, Grandori R, Ettrich R, Carey J (2009). "Structural organization of WrbA in apo- and holoprotein crystals." Biochim Biophys Acta 1794(9);1288-98. PMID: 19665595
Yang93: Yang W, Ni L, Somerville RL (1993). "A stationary-phase protein of Escherichia coli that affects the mode of association between the trp repressor protein and operator-bearing DNA." Proc Natl Acad Sci U S A 90(12);5796-800. PMID: 8516330
Zafar09: Zafar MN, Tasca F, Gorton L, Patridge EV, Ferry JG, Noll G (2009). "Tryptophan repressor-binding proteins from Escherichia coli and Archaeoglobus fulgidus as new catalysts for 1,4-dihydronicotinamide adenine dinucleotide-dependent amperometric biosensors and biofuel cells." Anal Chem 81(10);4082-8. PMID: 19438267
MendozaVargas09: Mendoza-Vargas A, Olvera L, Olvera M, Grande R, Vega-Alvarado L, Taboada B, Jimenez-Jacinto V, Salgado H, Juarez K, Contreras-Moreira B, Huerta AM, Collado-Vides J, Morett E (2009). "Genome-wide identification of transcription start sites, promoters and transcription factor binding sites in E. coli." PLoS One 4(10);e7526. PMID: 19838305
Ogasawara11: Ogasawara H, Yamamoto K, Ishihama A (2011). "Role of the Biofilm Master Regulator CsgD in Cross-Regulation between Biofilm Formation and Flagellar Synthesis." J Bacteriol 193(10);2587-97. PMID: 21421764
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