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Escherichia coli K-12 substr. MG1655 Protein: anaerobic nucleoside-triphosphate reductase activating system

Subunit composition of anaerobic nucleoside-triphosphate reductase activating system = [(NrdD)2][(NrdG)2][Fpr]
         ribonucleoside-triphosphate reductase = (NrdD)2
                 ribonucleoside-triphosphate reductase = NrdD
         ribonucleoside triphosphate reductase activase = (NrdG)2
         flavodoxin NADP+ reductase = Fpr (summary available)

Alternative forms of ribonucleoside-triphosphate reductase: oxidized ribonucleoside-triphosphate reductase

Summary:
The anaerobic nucleoside-triphosphate reductase activating system is composed of three enzymes and several compounds. Anaerobic nucleoside-triphosphate reductase is activated through the action of a specific activating enzyme, nucleoside-triphosphate reductase activase, flavodoxin NADP+ reductase, S-adenosylmethionine, flavodoxin and NADPH. All of these components form a multi-enzyme complex with the ribonucleoside reductase itself. [Bianchi93, Sun95]

Gene-Reaction Schematic

Gene-Reaction Schematic

Credits:
Last-Curated 24-Mar-2008 by Fulcher C, SRI International


Component enzyme of anaerobic nucleoside-triphosphate reductase activating system : ribonucleoside-triphosphate reductase

Gene: nrdD Accession Numbers: EG11417 (EcoCyc), b4238, ECK4233

Locations: cytosol

Subunit composition of ribonucleoside-triphosphate reductase = [NrdD]2
         ribonucleoside-triphosphate reductase = NrdD

Map Position: [4,458,545 <- 4,460,683] (96.1 centisomes, 346°)
Length: 2139 bp / 712 aa

Molecular Weight of Polypeptide: 80.023 kD (from nucleotide sequence)

pI: 6.87


GO Terms:
Biological Process:
Inferred from experimentGO:0015949 - nucleobase-containing small molecule interconversion [Garriga96]
Inferred by computational analysisGO:0006260 - DNA replication [GOA01a]
Inferred by computational analysisGO:0008152 - metabolic process [GOA01a]
Inferred by computational analysisGO:0055114 - oxidation-reduction process [UniProtGOA11a, GOA01a]
Molecular Function:
Inferred from experimentGO:0008270 - zinc ion binding [Luttringer09]
Inferred from experimentInferred by computational analysisGO:0008998 - ribonucleoside-triphosphate reductase activity [GOA01, GOA01a, Eliasson94]
Inferred from experimentGO:0042803 - protein homodimerization activity [Eliasson92]
Inferred from experimentGO:0051065 - CTP reductase activity [Eliasson92]
Inferred by computational analysisGO:0000166 - nucleotide binding [UniProtGOA11a]
Inferred by computational analysisGO:0003824 - catalytic activity [GOA01a]
Inferred by computational analysisGO:0005524 - ATP binding [UniProtGOA11a]
Inferred by computational analysisGO:0016491 - oxidoreductase activity [UniProtGOA11a]
Inferred by computational analysisGO:0046872 - metal ion binding [UniProtGOA11a]
Cellular Component:
Inferred from experimentGO:0031250 - anaerobic ribonucleoside-triphosphate reductase complex [Garriga96]
Inferred by computational analysisGO:0005829 - cytosol [DiazMejia09]

MultiFun Terms: metabolismcentral intermediary metabolism2'-deoxyribonucleotide metabolism

Unification Links: DIP:DIP-10358N, EcoliWiki:b4238, ModBase:P28903, Pride:P28903, Protein Model Portal:P28903, RefSeq:NP_418659, SMR:P28903, Swiss-Model:P28903, UniProt:P28903

Relationship Links: InterPro:IN-FAMILY:IPR001150, InterPro:IN-FAMILY:IPR005144, InterPro:IN-FAMILY:IPR012833, InterPro:IN-FAMILY:IPR019777, Pfam:IN-FAMILY:PF03477, Pfam:IN-FAMILY:PF13597, Prosite:IN-FAMILY:PS00850, Prosite:IN-FAMILY:PS51149, Prosite:IN-FAMILY:PS51161

Catalyzes:
dATP + an oxidized flavodoxin + H2O ← ATP + a reduced flavodoxin,
dGTP + an oxidized flavodoxin + H2O ← GTP + a reduced flavodoxin,
dUTP + an oxidized flavodoxin + H2O ← UTP + a reduced flavodoxin,
dCTP + an oxidized flavodoxin + H2O ← CTP + a reduced flavodoxin,
a reduced flavodoxin + a ribonucleoside triphosphate = an oxidized flavodoxin + a deoxyribonucleoside triphosphate + H2O

Summary:
The NrdD reductase is activated by the NrdG activase under anaerobic conditions and is inactivated by oxygen. The protein is highly sensitive to O2.

An nrdD null mutant does not grow under entirely anaerobic conditions, but grows under aerobic or microaerophilic conditions due to the activity of NrdA and/or NrdB [Garriga96].

Ribonucleotide reductase catalyzes the rate-limiting step in DNA biosynthesis. Its central role in DNA replication and repair makes its regulation important to ensure appropriate pools of deoxyribonucleotides for these processes. Three major classes I, II and III have been designated that share similar catalytic mechanisms. Enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium contain class Ia (encoded by nrdA and nrdB), class Ib (encoded by nrdE and nrdF) and class III (encoded by nrdD) enzymes. Class Ia and Ib enzymes are active under aerobic conditions, while class III enzymes are inactivated by oxygen and function under strictly anaerobic conditions. Although there are differences in structure and cofactor use, their catalytic mechanisms involve a transient cysteinyl radical at the active site that inititates ribonucleotide reduction. Regeneration of the enzymes is accomplished by corresponding reductive enzyme systems [Gon06] and discussed in [Torrents07].

This enzyme is a class III ribonucleotide reductase that is essential for anaerobic growth. In E. coli strain DHB4, construction of deletion mutants and analysis of their growth characteristics showed that the main ribonucleotide reductase activity in strict anaerobiosis is provided by the products of genes nrdD and nrdG. Under highly restricted oxygen condtions, the products of genes nrdA and nrdB can support some growth in a strain containing a deletion of nrdD and nrdG. Either thioredoxin reductase or glutathione reductase are required for anaerobic growth, suggesting that these reductive pathways contribute to enzyme activity [Gon06].

Essentiality data for nrdD knockouts:

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB enrichedYes 37 Aerobic 6.95   Yes [Gerdes03, Comment 1]
LB LennoxYes 37 Aerobic 7   Yes [Baba06, Comment 2]
M9 medium with 1% glycerolYes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 3]
MOPS medium with 0.4% glucoseYes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 2]
Yes [Feist07, Comment 4]

Component enzyme of anaerobic nucleoside-triphosphate reductase activating system : NrdG

Synonyms: yjgE

Gene: nrdG Accession Numbers: G812 (EcoCyc), b4237, ECK4232

Locations: cytosol

Subunit composition of ribonucleoside triphosphate reductase activase = [NrdG]2

Map Position: [4,457,923 <- 4,458,387] (96.08 centisomes, 346°)
Length: 465 bp / 154 aa

Molecular Weight of Polypeptide: 17.446 kD (from nucleotide sequence)


GO Terms:
Biological Process:
Inferred from experimentGO:0015949 - nucleobase-containing small molecule interconversion [Garriga96]
Inferred by computational analysisGO:0055114 - oxidation-reduction process [UniProtGOA11a, GOA01a]
Molecular Function:
Inferred from experimentInferred by computational analysisGO:0051536 - iron-sulfur cluster binding [UniProtGOA11a, Mulliez93]
Inferred from experimentInferred by computational analysisGO:0051539 - 4 iron, 4 sulfur cluster binding [UniProtGOA11a, GOA01a, Tamarit99]
Inferred by computational analysisGO:0003824 - catalytic activity [GOA01a]
Inferred by computational analysisGO:0016491 - oxidoreductase activity [UniProtGOA11a, GOA01a]
Inferred by computational analysisGO:0043365 - [formate-C-acetyltransferase]-activating enzyme activity [GOA01a]
Inferred by computational analysisGO:0046872 - metal ion binding [UniProtGOA11a]
Cellular Component:
Inferred from experimentGO:0031250 - anaerobic ribonucleoside-triphosphate reductase complex [Garriga96]
Inferred by computational analysisGO:0005737 - cytoplasm [UniProtGOA11, UniProtGOA11a, GOA01a]
Inferred by computational analysisGO:0005829 - cytosol [DiazMejia09]

MultiFun Terms: information transferprotein relatedposttranslational modification
metabolismcentral intermediary metabolismnucleotide and nucleoside conversions
regulationtype of regulationposttranscriptionalinhibition / activation of enzymes

Unification Links: DIP:DIP-48068N, EcoliWiki:b4237, PR:PRO_000023407, Protein Model Portal:P0A9N8, RefSeq:NP_418658, SMR:P0A9N8, UniProt:P0A9N8

Relationship Links: InterPro:IN-FAMILY:IPR001989, InterPro:IN-FAMILY:IPR012837, InterPro:IN-FAMILY:IPR013785, Pfam:IN-FAMILY:PF13353, Prosite:IN-FAMILY:PS01087

Catalyzes:
S-adenosyl-L-methionine + an inactive ribonucleoside triphosphate reductase ↔ 5'-deoxyadenosine + an active ribonucleoside triphosphate reductase + L-methionine

Summary:
An nrdG null mutant does not grow under entirely anaerobic conditions, but grows under aerobic or microaerophilic conditions due to the activity of NrdA and/or NrdB [Garriga96].

The NrdG activase activates the NrdD reductase under anaerobic conditions.

Using site-directed mutagenesis, the participation of three cysteine residues in iron chelation in the (4Fe-4S) cluster of this enzyme was demonstrated, but a fourth ligand remained unidentified [Tamarit00].

Citations: [Fontecave02]

Gene Citations: [Zaslaver06]

Essentiality data for nrdG knockouts:

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB enrichedYes 37 Aerobic 6.95   Yes [Gerdes03, Comment 1]
LB LennoxYes 37 Aerobic 7   Yes [Baba06, Comment 2]
M9 medium with 1% glycerolYes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 3]
MOPS medium with 0.4% glucoseYes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 2]
Yes [Feist07, Comment 4]

Component enzyme of anaerobic nucleoside-triphosphate reductase activating system : Fpr

Synonyms: FlxR, MvrA, Fpr, dA1

Gene: fpr Accession Numbers: EG10628 (EcoCyc), b3924, ECK3916

Locations: cytosol

Sequence Length: 248 AAs

Molecular Weight: 27.751 kD (from nucleotide sequence)

Molecular Weight: 28.0 kD (experimental) [Truniger92]

pI: 6.57


GO Terms:
Biological Process:
Inferred from experimentGO:0006001 - fructose catabolic process [Kornberg86]
Inferred from experimentGO:0042493 - response to drug [Morimyo88]
Inferred by computational analysisGO:0055114 - oxidation-reduction process [UniProtGOA11a, GOA01a]
Molecular Function:
Inferred from experimentInferred by computational analysisGO:0004324 - ferredoxin-NADP+ reductase activity [GOA01, Kornberg86, Bianchi93]
Inferred from experimentInferred by computational analysisGO:0016491 - oxidoreductase activity [UniProtGOA11a, GOA01a, Kornberg86]
Inferred from experimentGO:0071949 - FAD binding [Ingelman97]
Cellular Component:
Inferred from experimentGO:0005737 - cytoplasm [Kornberg86]
Inferred from experimentInferred by computational analysisGO:0005829 - cytosol [DiazMejia09, Ishihama08]

MultiFun Terms: cell processesprotectiondetoxification
metabolismcentral intermediary metabolismunassigned reversible reactions

Unification Links: EcoliWiki:b3924, ModBase:P28861, PR:PRO_000022684, Pride:P28861, Protein Model Portal:P28861, RefSeq:NP_418359, SMR:P28861, UniProt:P28861

Relationship Links: InterPro:IN-FAMILY:IPR001433, InterPro:IN-FAMILY:IPR008333, InterPro:IN-FAMILY:IPR017927, InterPro:IN-FAMILY:IPR017938, PDB:Structure:1FDR, PDB:Structure:2XNJ, Pfam:IN-FAMILY:PF00175, Pfam:IN-FAMILY:PF00970, Prosite:IN-FAMILY:PS51384

Catalyzes:
an oxidized flavodoxin + NADPH + H+ = a reduced flavodoxin + NADP+

Summary:
Fpr: ferredoxin NADP+ reductase [Bianchi93]

Citations: [Eliasson92]

Essentiality data for fpr knockouts:

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB enrichedYes 37 Aerobic 6.95   Yes [Gerdes03, Comment 1]
LB LennoxYes 37 Aerobic 7   Yes [Baba06, Comment 2]
M9 medium with 1% glycerolYes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 3]
MOPS medium with 0.4% glucoseYes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 2]
Yes [Feist07, Comment 4]

References

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

Bianchi93: Bianchi V, Reichard P, Eliasson R, Pontis E, Krook M, Jornvall H, Haggard-Ljungquist E (1993). "Escherichia coli ferredoxin NADP+ reductase: activation of E. coli anaerobic ribonucleotide reduction, cloning of the gene (fpr), and overexpression of the protein." J Bacteriol 1993;175(6);1590-5. PMID: 8449868

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

Eliasson92: Eliasson R, Pontis E, Fontecave M, Gerez C, Harder J, Jornvall H, Krook M, Reichard P (1992). "Characterization of components of the anaerobic ribonucleotide reductase system from Escherichia coli." J Biol Chem 267(35);25541-7. PMID: 1460049

Eliasson94: Eliasson R, Pontis E, Sun X, Reichard P (1994). "Allosteric control of the substrate specificity of the anaerobic ribonucleotide reductase from Escherichia coli." J Biol Chem 269(42);26052-7. PMID: 7929317

Feist07: Feist AM, Henry CS, Reed JL, Krummenacker M, Joyce AR, Karp PD, Broadbelt LJ, Hatzimanikatis V, Palsson BO (2007). "A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information." Mol Syst Biol 3;121. PMID: 17593909

Fontecave02: Fontecave M, Mulliez E, Logan DT (2002). "Deoxyribonucleotide synthesis in anaerobic microorganisms: the class III ribonucleotide reductase." Prog Nucleic Acid Res Mol Biol 2002;72;95-127. PMID: 12206460

Garriga96: Garriga X, Eliasson R, Torrents E, Jordan A, Barbe J, Gibert I, Reichard P (1996). "nrdD and nrdG genes are essential for strict anaerobic growth of Escherichia coli." Biochem Biophys Res Commun 1996;229(1);189-92. PMID: 8954104

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."

Gon06: Gon S, Faulkner MJ, Beckwith J (2006). "In vivo requirement for glutaredoxins and thioredoxins in the reduction of the ribonucleotide reductases of Escherichia coli." Antioxid Redox Signal 8(5-6);735-42. PMID: 16771665

Ingelman97: Ingelman M, Bianchi V, Eklund H (1997). "The three-dimensional structure of flavodoxin reductase from Escherichia coli at 1.7 A resolution." J Mol Biol 268(1);147-57. PMID: 9149148

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

Kornberg86: Kornberg H (1986). "The roles of HPr and FPr in the utilization of fructose by Escherichia coli." FEBS Lett 194(1);12-5. PMID: 3510127

Luttringer09: Luttringer F, Mulliez E, Dublet B, Lemaire D, Fontecave M (2009). "The Zn center of the anaerobic ribonucleotide reductase from E. coli." J Biol Inorg Chem 14(6);923-33. PMID: 19381696

Morimyo88: Morimyo M (1988). "Isolation and characterization of methyl viologen-sensitive mutants of Escherichia coli K-12." J Bacteriol 170(5);2136-42. PMID: 2834327

Mulliez93: Mulliez E, Fontecave M, Gaillard J, Reichard P (1993). "An iron-sulfur center and a free radical in the active anaerobic ribonucleotide reductase of Escherichia coli." J Biol Chem 1993;268(4);2296-9. PMID: 8381402

Sun95: Sun X, Eliasson R, Pontis E, Andersson J, Buist G, Sjoberg BM, Reichard P (1995). "Generation of the glycyl radical of the anaerobic Escherichia coli ribonucleotide reductase requires a specific activating enzyme." J Biol Chem 1995;270(6);2443-6. PMID: 7852304

Tamarit00: Tamarit J, Gerez C, Meier C, Mulliez E, Trautwein A, Fontecave M (2000). "The activating component of the anaerobic ribonucleotide reductase from Escherichia coli. An iron-sulfur center with only three cysteines." J Biol Chem 275(21);15669-75. PMID: 10821845

Tamarit99: Tamarit J, Mulliez E, Meier C, Trautwein A, Fontecave M (1999). "The anaerobic ribonucleotide reductase from Escherichia coli. The small protein is an activating enzyme containing a [4fe-4s](2+) center." J Biol Chem 1999;274(44);31291-6. PMID: 10531327

Torrents07: Torrents E, Grinberg I, Gorovitz-Harris B, Lundstrom H, Borovok I, Aharonowitz Y, Sjoberg BM, Cohen G (2007). "NrdR controls differential expression of the Escherichia coli ribonucleotide reductase genes." J Bacteriol 189(14);5012-21. PMID: 17496099

Truniger92: Truniger V, Boos W, Sweet G (1992). "Molecular analysis of the glpFKX regions of Escherichia coli and Shigella flexneri." J Bacteriol 174(21);6981-91. PMID: 1400248

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

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

Zaslaver06: Zaslaver A, Bren A, Ronen M, Itzkovitz S, Kikoin I, Shavit S, Liebermeister W, Surette MG, Alon U (2006). "A comprehensive library of fluorescent transcriptional reporters for Escherichia coli." Nat Methods 3(8);623-8. PMID: 16862137


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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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