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Escherichia coli K-12 substr. MG1655 Enzyme: NAD(P)H nitroreductase NfsB



Gene: nfsB Accession Numbers: EG50005 (EcoCyc), b0578, ECK0570

Synonyms: ntr, dprA, nfnB

Regulation Summary Diagram: ?

Subunit composition of NAD(P)H nitroreductase NfsB = [NfsB]2
         NfsB monomer = NfsB

Summary:
The nfsB-encoded nitroreductase is the minor oxygen-insensitive nitroreductase present in E. coli K-12. NfsB reduces a broad range of nitroaromatic compounds [Zenno96], including the antibiotics nitrofurazone and nitrofurantoin [McCalla78, Whiteway98, Bryant81]. NfsB is a flavin mononucleotide (FMN) containing protein and uses both NADH and NADPH as a source of reducing equivalent [Bryant81, Zenno96a, Lovering01].

Purifed NfsB has dihyropteridine reductase (DHPR) activity [Vasudevan88, Vasudevan92], pterin-independent NAD(P)H oxidoreductase activity with potassium ferricyanide as the electron acceptor [Vasudevan88] and dihyrofolate reductase activity [Vasudevan92].

NfsB forms a homodimer with subunit molecular mass of 27 kDa [Vasudevan88], or 23.9 kDa [Zenno96a]. NfsB is a monomer with a molecular mass of 25.7 kDa [Vasudevan92]. The crystal structure of dimeric NfsB in complex with nicotinic acid has been resolved at a resolution of 1.7 - 2.4 Å [Lovering01]. FAD can substitute for FMN as an effective prosthetic group [Zenno96a].

NfsB catalyses two successive, two-electron transfer reactions to reduce nitrosoaromatics to their hydroxylamine derivatives [Race05].

Bacterial nitroreductases such as NfsB have been used as prodrug activators for chemotherapy [Roldan08, Christofferson09].

Aspartate 37 has been implicated in binding the NADH cofactor [Grimshaw92].

NfsB: "nitrofuran sensitivity B" [McCalla78]

Citations: [Sastry84, Breeze83]

Locations: cytosol, membrane

Map Position: [603,994 <- 604,647] (13.02 centisomes)
Length: 654 bp / 217 aa

Molecular Weight of Polypeptide: 23.905 kD (from nucleotide sequence)

pI: 6.36

Unification Links: ASAP:ABE-0001981 , DIP:DIP-10330N , EchoBASE:EB4146 , EcoGene:EG20151 , EcoliWiki:b0578 , ModBase:P38489 , OU-Microarray:b0578 , PortEco:nfsB , PR:PRO_000023373 , Pride:P38489 , Protein Model Portal:P38489 , RefSeq:NP_415110 , RegulonDB:EG50005 , SMR:P38489 , String:511145.b0578 , UniProt:P38489

Relationship Links: InterPro:IN-FAMILY:IPR000415 , PDB:Structure:1DS7 , PDB:Structure:1ICR , PDB:Structure:1ICU , PDB:Structure:1ICV , PDB:Structure:1IDT , PDB:Structure:1OO5 , PDB:Structure:1OO6 , PDB:Structure:1OON , PDB:Structure:1OOQ , PDB:Structure:1YKI , PDB:Structure:1YLR , PDB:Structure:1YLU , Pfam:IN-FAMILY:PF00881

In Paralogous Gene Group: 157 (2 members)

Gene-Reaction Schematic: ?

Genetic Regulation Schematic: ?

GO Terms:

Biological Process: GO:0055114 - oxidation-reduction process Inferred by computational analysis [UniProtGOA11a]
Molecular Function: GO:0004155 - 6,7-dihydropteridine reductase activity Inferred from experiment Inferred by computational analysis [GOA01, Vasudevan88]
GO:0010181 - FMN binding Inferred from experiment [Zenno96a, Lovering01]
GO:0018545 - NAD(P)H nitroreductase activity Inferred from experiment [McCalla78, Bryant81, Breeze83a]
GO:0042802 - identical protein binding Inferred from experiment [Rajagopala14, Lasserre06]
GO:0042803 - protein homodimerization activity Inferred from experiment [Lovering01]
GO:0071949 - FAD binding Inferred from experiment [Vasudevan88]
GO:0016491 - oxidoreductase activity Inferred by computational analysis [UniProtGOA11a, GOA01a]
Cellular Component: GO:0005829 - cytosol Inferred from experiment [Ishihama08, LopezCampistrou05, Lasserre06]
GO:0016020 - membrane Inferred from experiment [Lasserre06]

MultiFun Terms: cell processes protection drug resistance/sensitivity
metabolism central intermediary metabolism unassigned reversible reactions

Essentiality data for nfsB 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]

Credits:
Last-Curated ? 22-Oct-2012 by Mackie A , Macquarie University


Enzymatic reaction of: NAD(P)H nitroreductase

EC Number: 1.7.1.-

an oxidized nitroaromatic compound + NAD(P)H <=> a reduced nitroaromatic compound + NAD(P)+

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.

The reaction is physiologically favored in the direction shown.

Alternative Substrates for an oxidized nitroaromatic compound: nitrofurazone [Valle12 ] , lawsone [Rau03 ] , 1,4-benzoquinone [Zenno96 ] , menadione [Zenno96 ] , ferricyanide [Zenno96 ]

Summary:
Kinetic parameters [Race05]
Km for NADH varies from 3-8 µM depending on nitro-substrate.
Km for nitrofurazone 160-170 µM.
Km for nitrofurantoin 160-190 µM.
Km for 2-nitrofuran 450-470 µM.


Enzymatic reaction of: dihydropteridine reductase (NAD(P)H nitroreductase NfsB)

Synonyms: NAD(P)H:6,7-dihydropteridine oxidoreductase, DHPR

EC Number: 1.5.1.34

a 5,6,7,8-tetrahydropteridine + NAD(P)+ <=> a 6,7-dihydropteridine + NAD(P)H + H+

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the Enzyme Commission system.

Reversibility of this reaction is unspecified.

Alternative Substrates for a 6,7-dihydropteridine [Vasudevan88 ]: cis-6-7-dimethyldihydro-(6H)-pterin , 6-methyldihydro-(6H)-pterin , dihydro-(6H)-monapterin , quinonoid dihydro-(6H)-biopterin , quinonoid dihydro-(6H)-neopterin

Summary:
Monapterin is the major pterin present in extracts prepared from E. coli lysates - traces of biopterin were also identified [Vasudevan88]. The physiological relevance of dihydropteridine reductase (DHPR) activity in E. coli is not clear.

Cofactors or Prosthetic Groups: FAD [Vasudevan88]

Inhibitors (Competitive): methotrexate [Comment 4]


Sequence Features

Feature Class Location Citations Comment
Sequence-Conflict 5
[Vasudevan88, UniProt10]
Alternate sequence: S → C; UniProt: (in Ref. 9; AA sequence);
Sequence-Conflict 10 -> 12
[Vasudevan88, UniProt10]
Alternate sequence: RHS → CIV; UniProt: (in Ref. 9; AA sequence);
Nucleotide-Phosphate-Binding-Region 10 -> 14
[UniProt10]
UniProt: FMN;
Sequence-Conflict 19
[Vasudevan88, UniProt10]
Alternate sequence: S → M; UniProt: (in Ref. 9; AA sequence);
Sequence-Conflict 21
[Anlezark92, UniProt10]
Alternate sequence: K → missing; UniProt: (in Ref. 7; AA sequence);
Sequence-Conflict 28
[Anlezark92, UniProt10]
Alternate sequence: E → D; UniProt: (in Ref. 7; AA sequence);
Amino-Acid-Sites-That-Bind 41
[UniProt10]
UniProt: NAD or NADP; via amide nitrogen;
Amino-Acid-Sites-That-Bind 71
[UniProt10]
UniProt: FMN;
Nucleotide-Phosphate-Binding-Region 153 -> 158
[UniProt10a]
UniProt: NAD or NADP; Non-Experimental Qualifier: by similarity;
Nucleotide-Phosphate-Binding-Region 165 -> 166
[UniProt10]
UniProt: FMN;
Sequence-Conflict 180
[Anlezark92, UniProt10]
Alternate sequence: E → I; UniProt: (in Ref. 7; AA sequence);
Nucleotide-Phosphate-Binding-Region 205 -> 207
[UniProt10]
UniProt: FMN;


Gene Local Context (not to scale): ?

Transcription Unit:

Notes:

History:
7/10/1998 (pkarp) Merged genes G557/b0578 and EG50005/nfnB
3/2/1998 (pkarp) Merged genes EG20151/nfnB and EG50005/EG20151
10/20/97 Gene b0578 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG20151; confirmed by SwissProt match.


References

Anlezark92: Anlezark GM, Melton RG, Sherwood RF, Coles B, Friedlos F, Knox RJ (1992). "The bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954)--I. Purification and properties of a nitroreductase enzyme from Escherichia coli--a potential enzyme for antibody-directed enzyme prodrug therapy (ADEPT)." Biochem Pharmacol 44(12);2289-95. PMID: 1472094

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

Breeze83: Breeze AS, Obaseiki-Ebor EE (1983). "Mutations to nitrofurantoin and nitrofurazone resistance in Escherichia coli K12." J Gen Microbiol 129(1);99-103. PMID: 6339681

Breeze83a: Breeze AS, Obaseiki-Ebor EE (1983). "Nitrofuran reductase activity in nitrofurantoin-resistant strains of Escherichia coli K12: some with chromosomally determined resistance and others carrying R-plasmids." J Antimicrob Chemother 12(6);543-7. PMID: 6363380

Bryant81: Bryant DW, McCalla DR, Leeksma M, Laneuville P (1981). "Type I nitroreductases of Escherichia coli." Can J Microbiol 27(1);81-6. PMID: 7011517

Christofferson09: Christofferson A, Wilkie J (2009). "Mechanism of CB1954 reduction by Escherichia coli nitroreductase." Biochem Soc Trans 37(Pt 2);413-8. PMID: 19290872

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, 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."

Grimshaw92: Grimshaw CE, Matthews DA, Varughese KI, Skinner M, Xuong NH, Bray T, Hoch J, Whiteley JM (1992). "Characterization and nucleotide binding properties of a mutant dihydropteridine reductase containing an aspartate 37-isoleucine replacement." J Biol Chem 1992;267(22);15334-9. PMID: 1639779

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

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

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

Lovering01: Lovering AL, Hyde EI, Searle PF, White SA (2001). "The structure of Escherichia coli nitroreductase complexed with nicotinic acid: three crystal forms at 1.7 A, 1.8 A and 2.4 A resolution." J Mol Biol 309(1);203-13. PMID: 11491290

McCalla78: McCalla DR, Kaiser C, Green MH (1978). "Genetics of nitrofurazone resistance in Escherichia coli." J Bacteriol 133(1);10-6. PMID: 338576

Race05: Race PR, Lovering AL, Green RM, Ossor A, White SA, Searle PF, Wrighton CJ, Hyde EI (2005). "Structural and mechanistic studies of Escherichia coli nitroreductase with the antibiotic nitrofurazone. Reversed binding orientations in different redox states of the enzyme." J Biol Chem 280(14);13256-64. PMID: 15684426

Rajagopala14: Rajagopala SV, Sikorski P, Kumar A, Mosca R, Vlasblom J, Arnold R, Franca-Koh J, Pakala SB, Phanse S, Ceol A, Hauser R, Siszler G, Wuchty S, Emili A, Babu M, Aloy P, Pieper R, Uetz P (2014). "The binary protein-protein interaction landscape of Escherichia coli." Nat Biotechnol 32(3);285-90. PMID: 24561554

Rau03: Rau J, Stolz A (2003). "Oxygen-insensitive nitroreductases NfsA and NfsB of Escherichia coli function under anaerobic conditions as lawsone-dependent Azo reductases." Appl Environ Microbiol 69(6);3448-55. PMID: 12788749

Roldan08: Roldan MD, Perez-Reinado E, Castillo F, Moreno-Vivian C (2008). "Reduction of polynitroaromatic compounds: the bacterial nitroreductases." FEMS Microbiol Rev 32(3);474-500. PMID: 18355273

Sastry84: Sastry SS, Jayaraman R (1984). "Nitrofurantoin-resistant mutants of Escherichia coli: isolation and mapping." Mol Gen Genet 196(2);379-80. PMID: 6387400

UniProt10: UniProt Consortium (2010). "UniProt version 2010-11 released on 2010-11-02 00:00:00." Database.

UniProt10a: UniProt Consortium (2010). "UniProt version 2010-07 released on 2010-06-15 00:00:00." Database.

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

Valle12: Valle A, Le Borgne S, Bolivar J, Cabrera G, Cantero D (2012). "Study of the role played by NfsA, NfsB nitroreductase and NemA flavin reductase from Escherichia coli in the conversion of ethyl 2-(2'-nitrophenoxy)acetate to 4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (D-DIBOA), a benzohydroxamic acid with interesting biological properties." Appl Microbiol Biotechnol 94(1);163-71. PMID: 22173483

Vasudevan88: Vasudevan SG, Shaw DC, Armarego WL (1988). "Dihydropteridine reductase from Escherichia coli." Biochem J 1988;255(2);581-8. PMID: 3060113

Vasudevan92: Vasudevan SG, Paal B, Armarego WL (1992). "Dihydropteridine reductase from Escherichia coli exhibits dihydrofolate reductase activity." Biol Chem Hoppe Seyler 373(10);1067-73. PMID: 1418677

Whiteway98: Whiteway J, Koziarz P, Veall J, Sandhu N, Kumar P, Hoecher B, Lambert IB (1998). "Oxygen-insensitive nitroreductases: analysis of the roles of nfsA and nfsB in development of resistance to 5-nitrofuran derivatives in Escherichia coli." J Bacteriol 180(21);5529-39. PMID: 9791100

Zenno96: Zenno S, Koike H, Tanokura M, Saigo K (1996). "Conversion of NfsB, a minor Escherichia coli nitroreductase, to a flavin reductase similar in biochemical properties to FRase I, the major flavin reductase in Vibrio fischeri, by a single amino acid substitution." J Bacteriol 178(15);4731-3. PMID: 8755909

Zenno96a: Zenno S, Koike H, Tanokura M, Saigo K (1996). "Gene cloning, purification, and characterization of NfsB, a minor oxygen-insensitive nitroreductase from Escherichia coli, similar in biochemical properties to FRase I, the major flavin reductase in Vibrio fischeri." J Biochem 120(4);736-44. PMID: 8947835

Other References Related to Gene Regulation

Barbosa02a: Barbosa TM, Levy SB (2002). "Activation of the Escherichia coli nfnB gene by MarA through a highly divergent marbox in a class II promoter." Mol Microbiol 45(1);191-202. PMID: 12100559

Martin02: Martin RG, Rosner JL (2002). "Genomics of the marA/soxS/rob regulon of Escherichia coli: identification of directly activated promoters by application of molecular genetics and informatics to microarray data." Mol Microbiol 44(6);1611-24. PMID: 12067348

Martin11a: Martin RG, Rosner JL (2011). "Promoter discrimination at class I MarA regulon promoters mediated by glutamic acid 89 of the MarA transcriptional activator of Escherichia coli." J Bacteriol 193(2);506-15. PMID: 21097628


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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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