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MetaCyc Enzyme: NADH:quinone reductase, FMN dependent / NADH-azoreductase

Gene: azoR Accession Numbers: G6731 (MetaCyc), b1412, ECK1405

Synonyms: acpD

Species: Escherichia coli K-12 substr. MG1655

Subunit composition of NADH:quinone reductase, FMN dependent / NADH-azoreductase = [AzoR]2
         NADH:quinone oxidoreductase, FMN-dependent = AzoR

Summary:
AzoR is an NADH:quinone oxidoreductase with a role in protection against thiol specific stress. Purified AzoR is active on a variety of electrophilic quinones [Liu09a]. An E. coli azoR::kan mutant has impaired growth in the presence of 2-methylhydroquinone, catechol and menadione and shows decreased levels of glutathione. Externally added glutathione can partially restore impaired growth of the azoR mutant caused by 2-methylhydroquinone [Liu09a].

Purified AzoR also has azoreductase activity [Nakanishi01]. Compounds containing azo bonds (N=N) have environmental and clinical significance and thus proteins with azordeuctase activity are subjects of experimental interest. AzoR was originally thought to be an acyl carrier protein phosphodiesterase [Fischl90] however, the overexpressed and purified protein has azoreductase activity, but not acyl carrier protein phosphodiesterase activity [Nakanishi01]. AzoR activity is FMN-dependent and requires 2 mol of NADH for the reduction of 1 mol of methyl red [Nakanishi01].

Crystal structures of AzoR in the oxidized and reduced forms have been solved [Ito06, Ito08]. The enzyme has structural similarity to flavodoxins; the FMN cofactors of each catalytic site are located at the dimer interface [Ito06]. The structure of the active site and the FMN cofactor differs between the oxidized and reduced forms [Ito08].

Transcription of azoR was induced by the presence of 0.5 mM 2-MHQ, 1 mM diamide, 6 mM catechol, 0.3 mM menadione,and 10 mM H2O2 [Liu09a].

Locations: cytosol

Map Position: [1,480,279 <- 1,480,884]

Molecular Weight of Polypeptide: 21.658 kD (from nucleotide sequence), 23.0 kD (experimental) [Nakanishi01 ]

Molecular Weight of Multimer: 42.0 kD (experimental) [Nakanishi01]

Unification Links: ASAP:ABE-0004712 , EchoBASE:EB2558 , EcoGene:EG12695 , EcoliWiki:B1412 , ModBase:P41407 , OU-Microarray:b1412 , PortEco:azoR , PR:PRO_000022195 , Pride:P41407 , Protein Model Portal:P41407 , RefSeq:NP_415930 , RegulonDB:G6731 , SMR:P41407 , String:511145.b1412 , UniProt:P41407

Relationship Links: InterPro:IN-FAMILY:IPR003680 , InterPro:IN-FAMILY:IPR023048 , Panther:IN-FAMILY:PTHR10204:SF4 , PDB:Structure:1TIK , PDB:Structure:1V4B , PDB:Structure:2D5I , PDB:Structure:2Z98 , PDB:Structure:2Z9B , PDB:Structure:2Z9C , PDB:Structure:2Z9D , Pfam:IN-FAMILY:PF02525

Gene-Reaction Schematic: ?

GO Terms:

Biological Process: GO:0006979 - response to oxidative stress Inferred from experiment [Nakanishi01]
GO:0055114 - oxidation-reduction process Inferred by computational analysis [UniProtGOA11a, GOA01a]
Molecular Function: GO:0010181 - FMN binding Inferred from experiment Inferred by computational analysis [GOA06, Nakanishi01]
GO:0016655 - oxidoreductase activity, acting on NAD(P)H, quinone or similar compound as acceptor Inferred from experiment [Liu09a]
GO:0050446 - azobenzene reductase activity Inferred from experiment [Nakanishi01]
GO:0008752 - FMN reductase activity Inferred by computational analysis [GOA06]
GO:0009055 - electron carrier activity Inferred by computational analysis [GOA06]
GO:0016491 - oxidoreductase activity Inferred by computational analysis [UniProtGOA11a]
GO:0016652 - oxidoreductase activity, acting on NAD(P)H, NAD(P) as acceptor Inferred by computational analysis [GOA06]
GO:0016661 - oxidoreductase activity, acting on other nitrogenous compounds as donors Inferred by computational analysis [GOA01a]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08, Zhang07]

MultiFun Terms: metabolism

Credits:
Created in EcoCyc 16-May-2008 by Keseler I , SRI International
Imported from EcoCyc 16-Sep-2014 by Paley S , SRI International


Enzymatic reaction of: NADH:quinone reductase, FMN dependent

EC Number: 1.6.5.-

a quinone + NADH <=> a semiquinone + NAD+

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction in which it was curated.

Reversibility of this reaction is unspecified.

Credits:
Imported from EcoCyc 16-Sep-2014 by Paley S , SRI International

Summary:
Km for 2-methyl-1,4-benzoquinone: 1704µM
Km for 2-hydroxy-1,4-napthoquinone: 87µM
Km for anthraquinone-2-sulfonate: 8.1µM
Km for anthraquinone-2, 6-disulfonate: 14.6µM


Enzymatic reaction of: NADH-azoreductase

EC Number: 1.7.1.-

methyl red + 2 NADH + 2 H+ <=> anthranilate + N,N'-dimethyl-p-phenylenediamine + 2 NAD+

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction in which it was curated.

Reversibility of this reaction is unspecified.

Note: The enzyme may catalyze this reaction in vitro, but this reaction is not considered to be physiologically relevant.

Alternative Substrates for methyl red: ethyl red [Nakanishi01 ] , menadione [Nakanishi01 ]

Credits:
Imported from EcoCyc 16-Sep-2014 by Paley S , SRI International

Summary:
FAD can substitute for FMN, but yielding lower catalytic activity. The enzyme is specific for NADH [Nakanishi01].

Cofactors or Prosthetic Groups: FMN [Nakanishi01]

Kinetic Parameters:

Substrate
Km (μM)
Citations
NADH
31.6
[Nakanishi01]
methyl red
17.9
[Nakanishi01]


Sequence Features

Feature Class Location Citations Comment
Cleavage-of-Initial-Methionine 1
[Fischl90, Nakanishi01]
 
Chain 2 -> 201
[UniProt09]
UniProt: FMN-dependent NADH-azoreductase;
Amino-Acid-Sites-That-Bind 10
[UniProt10a]
UniProt: FMN;
Nucleotide-Phosphate-Binding-Region 16 -> 18
[UniProt10a]
UniProt: FMN;
Sequence-Conflict 23 -> 35
[Kitakawa96, Moriya95, UniProt10a]
Alternate sequence: IILLNNGAKSTPR; UniProt: (in Ref. 1 and 5);
Nucleotide-Phosphate-Binding-Region 96 -> 99
[UniProt10a]
UniProt: FMN;
Nucleotide-Phosphate-Binding-Region 140 -> 145
[UniProt10a]
UniProt: FMN;

History:
Suzanne Paley on Mon Apr 14, 2003:
Comment from EcoGene: Azoreductase, NADH-dependent; previously thought to be AcpD, the acyl carrier protein phoshodiesterase. The real acpD gne and gene product now apparently need to be identified.
Markus Krummenacker on Tue Oct 14, 1997:
Gene object created from Blattner lab Genbank (v. M52) entry.


References

DiazMejia09: Diaz-Mejia JJ, Babu M, Emili A (2009). "Computational and experimental approaches to chart the Escherichia coli cell-envelope-associated proteome and interactome." FEMS Microbiol Rev 33(1);66-97. PMID: 19054114

Fischl90: Fischl AS, Kennedy EP (1990). "Isolation and properties of acyl carrier protein phosphodiesterase of Escherichia coli." J Bacteriol 172(9);5445-9. PMID: 2168383

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."

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

Ito06: Ito K, Nakanishi M, Lee WC, Sasaki H, Zenno S, Saigo K, Kitade Y, Tanokura M (2006). "Three-dimensional structure of AzoR from Escherichia coli. An oxidereductase conserved in microorganisms." J Biol Chem 281(29);20567-76. PMID: 16684776

Ito08: Ito K, Nakanishi M, Lee WC, Zhi Y, Sasaki H, Zenno S, Saigo K, Kitade Y, Tanokura M (2008). "Expansion of Substrate Specificity and Catalytic Mechanism of Azoreductase by X-ray Crystallography and Site-directed Mutagenesis." J Biol Chem 283(20);13889-13896. PMID: 18337254

Kitakawa96: Kitakawa M., Kasai H., Baba T., Honjo A., Isono K. (1996). "Nucleotide sequence of the replication terminus region of Escherichia coli." Data submission to EMBL/GenBank/DDBJ databases on 1996-05.

Liu09a: Liu G, Zhou J, Fu QS, Wang J (2009). "The Escherichia coli azoreductase AzoR Is involved in resistance to thiol-specific stress caused by electrophilic quinones." J Bacteriol 191(20);6394-400. PMID: 19666717

Moriya95: Moriya H, Kasai H, Isono K (1995). "Cloning and characterization of the hrpA gene in the terC region of Escherichia coli that is highly similar to the DEAH family RNA helicase genes of Saccharomyces cerevisiae." Nucleic Acids Res 23(4);595-8. PMID: 7899078

Nakanishi01: Nakanishi M, Yatome C, Ishida N, Kitade Y (2001). "Putative ACP phosphodiesterase gene (acpD) encodes an azoreductase." J Biol Chem 276(49);46394-9. PMID: 11583992

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.

UniProtGOA11a: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

Zhang07: Zhang N, Chen R, Young N, Wishart D, Winter P, Weiner JH, Li L (2007). "Comparison of SDS- and methanol-assisted protein solubilization and digestion methods for Escherichia coli membrane proteome analysis by 2-D LC-MS/MS." Proteomics 7(4);484-93. PMID: 17309111


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 SRI International Pathway Tools version 18.5 on Fri Dec 19, 2014, BIOCYC13B.