Escherichia coli K-12 substr. MG1655 Enzyme: N-ethylmaleimide reductase, FMN-linked

Gene: nemA Accession Numbers: G6890 (EcoCyc), b1650, ECK1646

Synonyms: ydhN

Regulation Summary Diagram: ?

Regulation summary diagram for nemA

N-ethylmaleimide reductase (NemA) is a member of the "old yellow enzyme" family of flavoproteins and catalyzes the reduction of N-ethylmaleimide (NEM) to N-ethylsuccinimide [Miura97, Miura97a]. NEM modifies the cysteine residues of cellular proteins and thus inhibits growth. NemA is probably involved in the degradation of toxic compounds and their reuse as a source of nitrogen [Umezawa08]. NemA is able to accept a variety of substrates, including the nitrate ester explosives pentaerythritol tetranitrate (PETN) and glycerol trinitrate (GTN) [Williams04b], quinones [Lee13e], and chromate [Robins13]. NemA can activate the nitroaromatic prodrug CB1954 [Prosser10].

E. coli K-12 AB1157 can utilize 2,4,6-trinitrotoluene (TNT) as a source of nitrogen. NemA together with NfsA and NfsB is responsible for this ability [GonzalezPerez07].

NemA activity is induced by menadione, dimethyl maleate and linoleic acid, possibly due to lipid peroxidation [Miura97]. Transcription of nemA is repressed by NemR, which is inactivated by alkylation [Umezawa08] or reversible oxidation of cysteine residues [Lee13e].

NemA: "N-ethylmaleimide reductase" [Miura97a]

Review: [Williams02a]

Gene Citations: [Ozyamak13]

Locations: cytosol

Map Position: [1,724,683 -> 1,725,780] (37.17 centisomes, 134°)
Length: 1098 bp / 365 aa

Molecular Weight of Polypeptide: 39.516 kD (from nucleotide sequence), 40.0 kD (experimental) [Miura97a ]

Unification Links: ASAP:ABE-0005516 , EchoBASE:EB3316 , EcoGene:EG13546 , EcoliWiki:b1650 , ModBase:P77258 , OU-Microarray:b1650 , PortEco:nemA , PR:PRO_000023369 , Pride:P77258 , Protein Model Portal:P77258 , RefSeq:NP_416167 , RegulonDB:G6890 , SMR:P77258 , String:511145.b1650 , Swiss-Model:P77258 , UniProt:P77258

Relationship Links: InterPro:IN-FAMILY:IPR001155 , InterPro:IN-FAMILY:IPR013785 , Pfam:IN-FAMILY:PF00724

In Paralogous Gene Group: 324 (2 members)

Gene-Reaction Schematic: ?

Gene-Reaction Schematic

Genetic Regulation Schematic: ?

Genetic regulation schematic for nemA

GO Terms:

Biological Process: GO:0006805 - xenobiotic metabolic process Inferred from experiment [Miura97a]
GO:0018937 - nitroglycerin metabolic process Inferred from experiment [Williams04b]
GO:0046256 - 2,4,6-trinitrotoluene catabolic process Inferred from experiment [GonzalezPerez07]
GO:0055114 - oxidation-reduction process Inferred by computational analysis [UniProtGOA11, GOA01]
Molecular Function: GO:0008748 - N-ethylmaleimide reductase activity Inferred from experiment [Miura97a]
GO:0010181 - FMN binding Inferred from experiment Inferred by computational analysis [GOA01, Prosser10]
GO:0034567 - chromate reductase activity Inferred from experiment [Robins13]
GO:0046857 - oxidoreductase activity, acting on other nitrogenous compounds as donors, with NAD or NADP as acceptor Inferred from experiment [Williams04b]
GO:0003824 - catalytic activity Inferred by computational analysis [GOA01]
GO:0016491 - oxidoreductase activity Inferred by computational analysis [UniProtGOA11, GOA01]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08, LopezCampistrou05]

MultiFun Terms: cell processes protection detoxification
metabolism central intermediary metabolism unassigned reversible reactions

Essentiality data for nemA 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]

Curated 11-May-2006 by Shearer A , SRI International
Last-Curated ? 28-Mar-2013 by Keseler I , SRI International

Enzymatic reaction of: N-ethylmaleimide reductase

EC Number: 1.3.1.-

N-ethylmaleimide + NADPH + H+ <=> N-ethylsuccinimide + NADP+

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. [Mizugaki81]

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

Alternative Substrates for N-ethylmaleimide: Cr6+ [Robins13 ] , menadione [Lee13e ] , ubiquinone-0 [Lee13e ] , 2,4,6-trinitrotoluene [GonzalezPerez07 ] , (E)-2-hexenal [Williams04b ] , pentaerythritol tetranitrate [Williams04b ] , nitroglycerin [vanDillewijn08 , Williams04b ]

Specific activity measured using trans-2-hexenal as a substrate and NADPH as co-substrate was approximately twice as high as with NADH as co-substrate [Williams04b]. A range of alternative substrates for NEM was tested by [Lee13e].

Using TNT as a substrate, the enzyme produces Meisenheimer dihydrate complex intermediates [vanDillewijn08].

Cofactors or Prosthetic Groups: FMN [Prosser10]

Inhibitors (Unknown Mechanism): p-hydroxymercuribenzoate [Mizugaki81]

Kinetic Parameters:

Km (μM)
kcat (sec-1)
kcat/Km (sec-1 μM-1)
Vmax (µmol mg-1 min-1)
Specific Activity (U/mg)

pH(opt): 7-8 [Mizugaki81]

Sequence Features

Protein sequence of N-ethylmaleimide reductase, FMN-linked with features indicated

Feature Class Location Citations Comment
Amino-Acid-Sites-That-Bind 101
UniProt: FMN; Non-Experimental Qualifier: by similarity;
Protein-Segment 182 -> 185
UniProt: Substrate-binding; Sequence Annotation Type: region of interest; Non-Experimental Qualifier: by similarity;
Active-Site 187
UniProt: Proton donor; Non-Experimental Qualifier: by similarity;
Amino-Acid-Sites-That-Bind 234
UniProt: FMN; Non-Experimental Qualifier: by similarity;
Amino-Acid-Sites-That-Bind 325
UniProt: FMN; Non-Experimental Qualifier: by similarity;

Gene Local Context (not to scale): ?

Gene local context diagram

Transcription Units:

Transcription-unit diagram

Transcription-unit diagram

Transcription-unit diagram

Transcription-unit diagram

Transcription-unit diagram


Markus Krummenacker on Tue Oct 14, 1997:
Gene object created from Blattner lab Genbank (v. M52) entry.


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

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

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."

GonzalezPerez07: Gonzalez-Perez MM, van Dillewijn P, Wittich RM, Ramos JL (2007). "Escherichia coli has multiple enzymes that attack TNT and release nitrogen for growth." Environ Microbiol 9(6);1535-40. PMID: 17504490

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

Lee13e: Lee C, Shin J, Park C (2013). "Novel regulatory system nemRA-gloA for electrophile reduction in Escherichia coli K-12." Mol Microbiol 88(2);395-412. PMID: 23506073

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

Miura97: Miura K, Tomioka Y, Hoshi Y, Suzuki H, Yonezawa M, Hishinuma T, Mizugaki M (1997). "The effects of unsaturated fatty acids, oxidizing agents and Michael reaction acceptors on the induction of N-ethylmaleimide reductase in Escherichia coli: possible application for drug design of chemoprotectors." Methods Find Exp Clin Pharmacol 19(3);147-51. PMID: 9203161

Miura97a: Miura K, Tomioka Y, Suzuki H, Yonezawa M, Hishinuma T, Mizugaki M (1997). "Molecular cloning of the nemA gene encoding N-ethylmaleimide reductase from Escherichia coli." Biol Pharm Bull 20(1);110-2. PMID: 9013822

Mizugaki81: Mizugaki M, Unuma T, Shiraishi T, Nishimaki T, Yamanaka H (1981). "Studies on the metabolism of unsaturated fatty acids. IV. N-Ethylmaleimide reducing activity in Escherichia coli K-12." Chem Pharm Bull (Tokyo) 29(2);570-3. PMID: 7023711

Ozyamak13: Ozyamak E, de Almeida C, de Moura AP, Miller S, Booth IR (2013). "Integrated stress response of Escherichia coli to methylglyoxal: transcriptional readthrough from the nemRA operon enhances protection through increased expression of glyoxalase I." Mol Microbiol 88(5);936-50. PMID: 23646895

Prosser10: Prosser GA, Copp JN, Syddall SP, Williams EM, Smaill JB, Wilson WR, Patterson AV, Ackerley DF (2010). "Discovery and evaluation of Escherichia coli nitroreductases that activate the anti-cancer prodrug CB1954." Biochem Pharmacol 79(5);678-87. PMID: 19852945

Robins13: Robins KJ, Hooks DO, Rehm BH, Ackerley DF (2013). "Escherichia coli NemA Is an Efficient Chromate Reductase That Can Be Biologically Immobilized to Provide a Cell Free System for Remediation of Hexavalent Chromium." PLoS One 8(3);e59200. PMID: 23527133

Umezawa08: Umezawa Y, Shimada T, Kori A, Yamada K, Ishihama A (2008). "Uncharacterized Transcription Factor YdhM is the Regulator of the nemA Gene Encoding N-ethylmaleimide (NEM) Reductase." J Bacteriol 190(17):5890-7. PMID: 18567656

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

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

vanDillewijn08: van Dillewijn P, Wittich RM, Caballero A, Ramos JL (2008). "Type II hydride transferases from different microorganisms yield nitrite and diarylamines from polynitroaromatic compounds." Appl Environ Microbiol 74(21);6820-3. PMID: 18791007

Williams02a: Williams RE, Bruce NC (2002). "'New uses for an Old Enzyme'--the Old Yellow Enzyme family of flavoenzymes." Microbiology 148(Pt 6);1607-14. PMID: 12055282

Williams04b: Williams RE, Rathbone DA, Scrutton NS, Bruce NC (2004). "Biotransformation of explosives by the old yellow enzyme family of flavoproteins." Appl Environ Microbiol 70(6);3566-74. PMID: 15184158

Other References Related to Gene Regulation

Gogol11: Gogol EB, Rhodius VA, Papenfort K, Vogel J, Gross CA (2011). "Small RNAs endow a transcriptional activator with essential repressor functions for single-tier control of a global stress regulon." Proc Natl Acad Sci U S A 108(31);12875-80. PMID: 21768388

Gray13: Gray MJ, Wholey WY, Parker BW, Kim M, Jakob U (2013). "NemR is a bleach-sensing transcription factor." J Biol Chem 288(19);13789-98. PMID: 23536188

Shimada08: Shimada T, Ishihama A, Busby SJ, Grainger DC (2008). "The Escherichia coli RutR transcription factor binds at targets within genes as well as intergenic regions." Nucleic Acids Res 36(12);3950-5. PMID: 18515344

Report Errors or Provide Feedback
Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
Page generated by SRI International Pathway Tools version 19.0 on Thu Oct 8, 2015, biocyc13.