Escherichia coli K-12 substr. MG1655 Enzyme: NAD(P)H:quinone oxidoreductase

Gene: wrbA Accession Numbers: EG11540 (EcoCyc), b1004, ECK0995

Regulation Summary Diagram: ?

Regulation summary diagram for wrbA

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 [Yang93a]. 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 [Yang93a]. The association between WrbA and TrpR observed by [Yang93a] may therefore be due to structural rather than functional reasons [Grandori98].

Expression of wrbA is increased during stationary phase and is RpoS-dependent [Yang93a, 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 [Liu04a].

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" [Yang93a]

Locations: cytosol, membrane

Map Position: [1,066,335 <- 1,066,931] (22.98 centisomes, 83°)
Length: 597 bp / 198 aa

Molecular Weight of Polypeptide: 20.846 kD (from nucleotide sequence), 21 kD (experimental) [Yang93a ]

pI: 5.7

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 , InterPro:IN-FAMILY:IPR029039 , 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

Gene-Reaction Schematic: ?

Gene-Reaction Schematic

Instance reactions of [an electron-transfer quinone[inner membrane] + NAD(P)H + H+ → an electron-transfer quinol[inner membrane] + NAD(P)+] (
i1: NADH + a ubiquinone[inner membrane] + H+ → NAD+ + an ubiquinol[inner membrane] (

i2: NADH + a menaquinone[inner membrane] + H+ → NAD+ + a menaquinol[inner membrane] (no EC#)

Genetic Regulation Schematic: ?

Genetic regulation schematic for wrbA

GO Terms:

Biological Process: GO:0006979 - response to oxidative stress Inferred from experiment [Lacour04]
GO:0045892 - negative regulation of transcription, DNA-templated Inferred by computational analysis [GOA01]
GO:0055114 - oxidation-reduction process Inferred by computational analysis [UniProtGOA11]
Molecular Function: GO:0003955 - NAD(P)H dehydrogenase (quinone) activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01a, Patridge06]
GO:0010181 - FMN binding Inferred from experiment Inferred by computational analysis [GOA01, Patridge06]
GO:0042802 - identical protein binding Inferred from experiment [Lasserre06]
GO:0000166 - nucleotide binding Inferred by computational analysis [UniProtGOA11]
GO:0016491 - oxidoreductase activity Inferred by computational analysis [UniProtGOA11, GOA01]
GO:0050660 - flavin adenine dinucleotide binding Inferred by computational analysis [GOA06]
GO:0050661 - NADP binding Inferred by computational analysis [GOA06]
GO:0051287 - NAD binding Inferred by computational analysis [GOA06]
Cellular Component: GO:0005829 - cytosol Inferred from experiment [Ishihama08, LopezCampistrou05, Patridge06]
GO:0016020 - membrane Inferred from experiment [Lasserre06]
GO:0005737 - cytoplasm


Essentiality data for wrbA knockouts: ?

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]

Created 17-Oct-2007 by Keseler I , SRI International
Last-Curated ? 19-Oct-2012 by Keseler I , SRI International

Enzymatic reaction of: NAD(P)H:quinone oxidoreductase

EC Number:

Transport reaction diagram for NAD(P)H:quinone oxidoreductase

Alternative Substrates for an electron-transfer quinone: menadione [Patridge06 ] , 2,3-dihydroxy-5-methyl-1,4-benzoquinone [Patridge06 ] , 1,4-naphthoquinone [Patridge06 ] , 1,4-benzoquinone [Patridge06 ]

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].

Cofactors or Prosthetic Groups: FMN [Patridge06]

Kinetic Parameters:

Km (μM)
an electron-transfer quinone

pH(opt): 6-8 [Patridge06]

Sequence Features

Protein sequence of NAD(P)H:quinone oxidoreductase with features indicated

Feature Class Location Attached Group Citations Comment
Cleavage-of-Initial-Methionine 1  
[Grandori98, Yang93a, Patridge06]
Chain 2 -> 198  
UniProt: Flavoprotein wrbA;
Conserved-Region 4 -> 189  
UniProt: Flavodoxin-like;
Nucleotide-Phosphate-Binding-Region 9 -> 14 FMN
[Wolfova09, Andrade07, UniProt15]
UniProt: FMN.
Amino-Acid-Sites-That-Bind 12  
UniProt: NAD.
N6-acetyllysine-Modification 50  
[Zhang09a, UniProt15]
UniProt: N6-acetyllysine.
Amino-Acid-Sites-That-Bind 51  
UniProt: NAD; via carbonyl oxygen.
Nucleotide-Phosphate-Binding-Region 77 -> 80 FMN
[Wolfova09, Andrade07, UniProt15]
UniProt: FMN.
Amino-Acid-Sites-That-Bind 98  
UniProt: Substrate.
Nucleotide-Phosphate-Binding-Region 112 -> 118 FMN
[Wolfova09, Andrade07, UniProt15]
UniProt: FMN.
Amino-Acid-Sites-That-Bind 133  
[Wolfova09, Andrade07, UniProt15]
UniProt: FMN.
Sequence-Conflict 142  
[Yang93a, UniProt10a]
UniProt: (in Ref. 1; AAA24759);

Gene Local Context (not to scale): ?

Gene local context diagram

Transcription Unit:

Transcription-unit diagram


10/20/97 Gene b1004 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG11540; confirmed by SwissProt match.


Andrade07: Andrade SL, Patridge EV, Ferry JG, Einsle O (2007). "Crystal structure of the NADH:quinone oxidoreductase WrbA from Escherichia coli." J Bacteriol 189(24);9101-7. PMID: 17951395

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

GOA01: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

GOA01a: GOA, MGI (2001). "Gene Ontology annotation based on Enzyme Commission mapping." Genomics 74;121-128.

GOA06: GOA, SIB (2006). "Electronic Gene Ontology annotations created by transferring manual GO annotations between orthologous microbial proteins."

Grandori94: Grandori R, Carey J (1994). "Six new candidate members of the alpha/beta twisted open-sheet family detected by sequence similarity to flavodoxin." Protein Sci 3(12);2185-93. PMID: 7756978

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

Ishihama08: Ishihama Y, Schmidt T, Rappsilber J, Mann M, Hartl FU, Kerner MJ, Frishman D (2008). "Protein abundance profiling of the Escherichia coli cytosol." BMC Genomics 9;102. PMID: 18304323

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

Lelong07: Lelong C, Aguiluz K, Luche S, Kuhn L, Garin J, Rabilloud T, Geiselmann J (2007). "The Crl-RpoS regulon of Escherichia coli." Mol Cell Proteomics 6(4);648-59. PMID: 17224607

Liu04a: 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

Patridge06: Patridge EV, Ferry JG (2006). "WrbA from Escherichia coli and Archaeoglobus fulgidus is an NAD(P)H:quinone oxidoreductase." J Bacteriol 188(10);3498-506. PMID: 16672604

Tjaden06: Tjaden B, Goodwin SS, Opdyke JA, Guillier M, Fu DX, Gottesman S, Storz G (2006). "Target prediction for small, noncoding RNAs in bacteria." Nucleic Acids Res 34(9);2791-802. PMID: 16717284

UniProt09: UniProt Consortium (2009). "UniProt version 15.8 released on 2009-10-01 00:00:00." Database.

UniProt10a: UniProt Consortium (2010). "UniProt version 2010-11 released on 2010-11-02 00:00:00." Database.

UniProt15: UniProt Consortium (2015). "UniProt version 2015-01 released on 2015-01-16 00:00:00." Database.

UniProtGOA11: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

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

Yang93a: 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

Zhang09a: Zhang J, Sprung R, Pei J, Tan X, Kim S, Zhu H, Liu CF, Grishin NV, Zhao Y (2009). "Lysine acetylation is a highly abundant and evolutionarily conserved modification in Escherichia coli." Mol Cell Proteomics 8(2);215-25. PMID: 18723842

Other References Related to Gene Regulation

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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Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
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