MetaCyc Enzyme: NAD(P)H:quinone oxidoreductase

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

Species: Escherichia coli K-12 substr. MG1655

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 [Liu04b].

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: [1,067,112 <- 1,067,708]

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:PF03358, Prosite:IN-FAMILY:PS50902

Gene-Reaction Schematic

Expand/Contract the Schematic connections:

Gene-Reaction Schematic

GO Terms:
Biological Process:
Inferred from experimentGO:0006979 - response to oxidative stress [Lacour04]
Inferred by computational analysisGO:0045892 - negative regulation of transcription, DNA-templated [GOA01a]
Inferred by computational analysisGO:0055114 - oxidation-reduction process [UniProtGOA11a]
Molecular Function:
Inferred from experimentInferred by computational analysisGO:0003955 - NAD(P)H dehydrogenase (quinone) activity [GOA06, GOA01, Patridge06]
Inferred from experimentInferred by computational analysisGO:0010181 - FMN binding [GOA01a, Patridge06]
Inferred from experimentGO:0042802 - identical protein binding [Lasserre06]
Inferred by computational analysisGO:0000166 - nucleotide binding [UniProtGOA11a]
Inferred by computational analysisGO:0016491 - oxidoreductase activity [UniProtGOA11a, GOA01a]
Inferred by computational analysisGO:0050660 - flavin adenine dinucleotide binding [GOA06]
Inferred by computational analysisGO:0050661 - NADP binding [GOA06]
Inferred by computational analysisGO:0051287 - NAD binding [GOA06]
Cellular Component:
Inferred from experimentGO:0005829 - cytosol [Ishihama08, LopezCampistrou05, Patridge06]
Inferred from experimentGO:0016020 - membrane [Lasserre06]


Created in EcoCyc 17-Oct-2007 by Keseler I, SRI International
Imported from EcoCyc 15-Mar-2016 by Paley S, SRI International

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

Inferred from experiment

EC Number:

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

Alternative Substrates for NAD(P)H: NADH [Patridge06], NADPH [Patridge06]
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]

Imported from EcoCyc 15-Mar-2016 by Paley S, SRI International

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:
Substrate Km (μM) kcat (sec-1) kcat/Km (sec-1 μM-1) Citations
1,4-benzoquinone 5.8 370.0 63.79 [Patridge06]
NADH 14.0 8.9 0.64 [Patridge06]
NADPH 170.0 6.0 0.035 [Patridge06]

pH(opt): 6-8 [Patridge06]

Sequence Features

Feature Class Location Attached Group Citations Comment
Cleavage-of-Initial-Methionine 1  
Chain 2 -> 198  
Author statement[UniProt15]
UniProt: NAD(P)H dehydrogenase (quinone).
Conserved-Region 4 -> 189  
Author statement[UniProt15]
UniProt: Flavodoxin-like.
Nucleotide-Phosphate-Binding-Region 9 -> 14 FMN
Inferred from experiment[Wolfova09, Andrade07]
UniProt: FMN.
Amino-Acid-Sites-That-Bind 12  
Author statement[UniProt15]
UniProt: NAD.
N6-acetyllysine-Modification 50  
Inferred from experiment[Zhang09c]
UniProt: N6-acetyllysine.
Amino-Acid-Sites-That-Bind 51  
Author statement[UniProt15]
UniProt: NAD; via carbonyl oxygen.
Nucleotide-Phosphate-Binding-Region 77 -> 80 FMN
Inferred from experiment[Wolfova09, Andrade07]
UniProt: FMN.
Amino-Acid-Sites-That-Bind 98  
Author statement[UniProt15]
UniProt: Substrate.
Nucleotide-Phosphate-Binding-Region 112 -> 118 FMN
Inferred from experiment[Wolfova09, Andrade07]
UniProt: FMN.
Amino-Acid-Sites-That-Bind 133  
Inferred from experiment[Wolfova09, Andrade07]
UniProt: FMN.
Sequence-Conflict 142  
Inferred by curator[Yang93a, UniProt15]
UniProt: (in Ref. 1; AAA24759).

Sequence Pfam Features

Feature Class Location Citations Comment
Pfam PF03358 13 -> 143
Inferred by computational analysis[Finn14]
FMN_red : NADPH-dependent FMN reductase


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

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

Finn14: Finn RD, Bateman A, Clements J, Coggill P, Eberhardt RY, Eddy SR, Heger A, Hetherington K, Holm L, Mistry J, Sonnhammer EL, Tate J, Punta M (2014). "Pfam: the protein families database." Nucleic Acids Res 42(Database issue);D222-30. PMID: 24288371

GOA01: GOA, MGI (2001). "Gene Ontology annotation based on Enzyme Commission mapping." Genomics 74;121-128.

GOA01a: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

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

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

Liu04b: 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

UniProt15: UniProt Consortium (2015). "UniProt version 2015-08 released on 2015-07-22." Database.

UniProtGOA11a: 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

Zhang09c: 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

Report Errors or Provide Feedback
Please cite the following article in publications resulting from the use of MetaCyc: Caspi et al, Nucleic Acids Research 42:D459-D471 2014
Page generated by Pathway Tools version 20.0 (software by SRI International) on Fri May 6, 2016, BIOCYC13B.