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Escherichia coli K-12 substr. MG1655 Enzyme: quinolinate synthase



Gene: nadA Accession Numbers: EG10630 (EcoCyc), b0750, ECK0739

Synonyms: nicA

Regulation Summary Diagram: ?

Subunit composition of quinolinate synthase = [NadA]2

Summary:
Quinolinate synthase catalyzes the second reaction in the de novo biosynthesis of NAD from aspartate, the condensation of iminosuccinate with dihydroxyacetone phosphate to form quinolinate.

NadA was shown to contain an oxygen-sensitive [4Fe-4S] cluster that is required for its activity [Cicchillo05, Ollagnierde05], explaining the NAD auxotrophy of an iscS mutant. Reaction mechanisms involving the [4Fe-4S] cluster have been proposed [Cicchillo05]. The conserved C113, C200 and C297 residues are involved in [4Fe-4S] cluster binding [Rousset08]. The C291 and C294 residues appear to undergo reversible disulfide bond formation that regulates the activity of the enzyme [Saunders08, Rousset08]. The midpoint potential of -264 mV is similar to that of thioredoxin and of the cytosol [Saunders08].

NadA: "NAD biosynthesis" [Tritz70]

Locations: cytosol

Map Position: [781,308 -> 782,351] (16.84 centisomes)
Length: 1044 bp / 347 aa

Molecular Weight of Polypeptide: 38.241 kD (from nucleotide sequence), 38.2 kD (experimental) [Ceciliani00 ]

Molecular Weight of Multimer: 80.0 kD (experimental) [Ollagnierde05]

pI: 6.02

Unification Links: ASAP:ABE-0002541 , CGSC:467 , DIP:DIP-1027N , EchoBASE:EB0624 , EcoGene:EG10630 , EcoliWiki:b0750 , OU-Microarray:b0750 , PortEco:nadA , PR:PRO_000023332 , Pride:P11458 , Protein Model Portal:P11458 , RefSeq:NP_415271 , RegulonDB:EG10630 , SMR:P11458 , String:511145.b0750 , UniProt:P11458

Relationship Links: InterPro:IN-FAMILY:IPR003473 , InterPro:IN-FAMILY:IPR023513 , Pfam:IN-FAMILY:PF02445

Gene-Reaction Schematic: ?

Genetic Regulation Schematic: ?

GO Terms:

Biological Process: GO:0019805 - quinolinate biosynthetic process Inferred by computational analysis Inferred from experiment [Flachmann88, Ollagnierde05, GOA06, GOA01]
GO:0034628 - 'de novo' NAD biosynthetic process from aspartate Inferred from experiment [Ollagnierde05, Kerr73, Taylor67a]
GO:0009435 - NAD biosynthetic process Inferred by computational analysis [UniProtGOA12, GOA06, GOA01]
GO:0019363 - pyridine nucleotide biosynthetic process Inferred by computational analysis [UniProtGOA11]
Molecular Function: GO:0008987 - quinolinate synthetase A activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01, Ollagnierde05, Flachmann88]
GO:0051539 - 4 iron, 4 sulfur cluster binding Inferred from experiment Inferred by computational analysis [UniProtGOA11, GOA01, Rousset08, Cicchillo05]
GO:0016740 - transferase activity Inferred by computational analysis [UniProtGOA11]
GO:0016765 - transferase activity, transferring alkyl or aryl (other than methyl) groups Inferred by computational analysis [GOA01]
GO:0046872 - metal ion binding Inferred by computational analysis [UniProtGOA11]
GO:0051536 - iron-sulfur cluster binding Inferred by computational analysis [UniProtGOA11]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08, LopezCampistrou05]
GO:0005737 - cytoplasm Inferred by computational analysis [UniProtGOA11a, UniProtGOA11, GOA06]

MultiFun Terms: metabolism biosynthesis of building blocks cofactors, small molecule carriers nicotinamide adenine dinucleotide

Essentiality data for nadA 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 0.4% glucose No 37 Aerobic 7.2 0.27 No [Patrick07, Comment 3]
M9 medium with 1% glycerol No 37 Aerobic 7.2 0.35 No [Joyce06]
MOPS medium with 0.4% glucose Indeterminate 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 2]
No [Feist07, Comment 4]

Credits:
Created 29-Jul-2008 by Keseler I , SRI International
Last-Curated ? 19-Aug-2008 by Keseler I , SRI International


Enzymatic reaction of: quinolinate synthase

EC Number: 2.5.1.72

α-iminosuccinate + dihydroxyacetone phosphate <=> quinolinate + phosphate + 2 H2O

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.

In Pathways: aspartate superpathway , NAD biosynthesis I (from aspartate)

Cofactors or Prosthetic Groups: a [4Fe-4S] iron-sulfur cluster [Cicchillo05, Ollagnierde05]

Inhibitors (Unknown Mechanism): oxygen [DraczynskaLusia92, Gardner91]

Kinetic Parameters:

Substrate
Km (μM)
Citations
dihydroxyacetone phosphate
200.0
[Suzuki73]

pH(opt): 8-8.5 [Suzuki73]


Sequence Features

Feature Class Location Citations Comment
Sequence-Conflict 35 -> 36
[Flachmann88, UniProt10a]
Alternate sequence: RL → LR; UniProt: (in Ref. 1; CAA31216);
Amino-Acid-Sites-That-Bind 47
[UniProt10]
UniProt: Iminoaspartate; Non-Experimental Qualifier: by similarity;
Amino-Acid-Sites-That-Bind 68
[UniProt10]
UniProt: Iminoaspartate; via amide nitrogen; Non-Experimental Qualifier: by similarity;
Amino-Acid-Sites-That-Bind 139
[UniProt10]
UniProt: Iminoaspartate; Non-Experimental Qualifier: by similarity;
Sequence-Conflict 153
[Flachmann88, UniProt10a]
Alternate sequence: V → G; UniProt: (in Ref. 1; CAA31216);
Amino-Acid-Sites-That-Bind 156
[UniProt10]
UniProt: Iminoaspartate; Non-Experimental Qualifier: by similarity;
Sequence-Conflict 170
[Flachmann88, UniProt10a]
Alternate sequence: G → C; UniProt: (in Ref. 1; CAA31216);
Amino-Acid-Sites-That-Bind 226
[UniProt10]
UniProt: Iminoaspartate; Non-Experimental Qualifier: by similarity;
Amino-Acid-Sites-That-Bind 243
[UniProt10]
UniProt: Iminoaspartate; Non-Experimental Qualifier: by similarity;
Mutagenesis-Variant 291
[Saunders08, UniProt11]
Alternate sequence: C → S; UniProt: Activity is 3.3-fold lower under oxic conditions than under anoxic conditions.
Disulfide-Bond-Site 294, 291
[Saunders08, Rousset08]
 
Mutagenesis-Variant 294
[Saunders08, UniProt11]
Alternate sequence: C → S; UniProt: Activity is 13-fold lower under oxic conditions than under anoxic conditions.
Sequence-Conflict 329
[Flachmann88, UniProt10a]
Alternate sequence: E → R; UniProt: (in Ref. 1; CAA31216);


Gene Local Context (not to scale): ?

Transcription Unit:

Notes:

History:
10/20/97 Gene b0750 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10630; confirmed by SwissProt match.


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

Ceciliani00: Ceciliani F, Caramori T, Ronchi S, Tedeschi G, Mortarino M, Galizzi A (2000). "Cloning, overexpression, and purification of Escherichia coli quinolinate synthetase." Protein Expr Purif 2000;18(1);64-70. PMID: 10648170

Cicchillo05: Cicchillo RM, Tu L, Stromberg JA, Hoffart LM, Krebs C, Booker SJ (2005). "Escherichia coli quinolinate synthetase does indeed harbor a [4Fe-4S] cluster." J Am Chem Soc 127(20);7310-1. PMID: 15898769

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

DraczynskaLusia92: Draczynska-Lusiak B, Brown OR (1992). "Protein A of quinolinate synthetase is the site of oxygen poisoning of pyridine nucleotide coenzyme synthesis in Escherichia coli." Free Radic Biol Med 13(6);689-93. PMID: 1459486

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

Flachmann88: Flachmann R, Kunz N, Seifert J, Gutlich M, Wientjes FJ, Laufer A, Gassen HG (1988). "Molecular biology of pyridine nucleotide biosynthesis in Escherichia coli. Cloning and characterization of quinolinate synthesis genes nadA and nadB." Eur J Biochem 1988;175(2);221-8. PMID: 2841129

Gardner91: Gardner PR, Fridovich I (1991). "Quinolinate synthetase: the oxygen-sensitive site of de novo NAD(P)+ biosynthesis." Arch Biochem Biophys 284(1);106-11. PMID: 1846509

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."

GOA06: GOA, SIB (2006). "Electronic Gene Ontology annotations created by transferring manual GO annotations between orthologous microbial proteins."

Griffith75: Griffith GR, Chandler JL, Gholson RK (1975). "Studies on the de novo biosynthesis of NAD in Escherichia coli. The separation of the nadB gene product from the nadA gene product and its purification." Eur J Biochem 54(1);239-45. PMID: 238844

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

Kerr73: Kerr TJ, Tritz GJ (1973). "Cross-feeding of Escherichia coli mutants defective in the biosynthesis of nicotinamide adenine dinucleotide." J Bacteriol 115(3);982-6. PMID: 4353874

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

Nasu82: Nasu S, Wicks FD, Gholson RK (1982). "The mammalian enzyme which replaces B protein of E. coli quinolinate synthetase is D-aspartate oxidase." Biochim Biophys Acta 704(2);240-52. PMID: 7049247

Ollagnierde05: Ollagnier-de Choudens S, Loiseau L, Sanakis Y, Barras F, Fontecave M (2005). "Quinolinate synthetase, an iron-sulfur enzyme in NAD biosynthesis." FEBS Lett 579(17);3737-43. PMID: 15967443

Patrick07: Patrick WM, Quandt EM, Swartzlander DB, Matsumura I (2007). "Multicopy suppression underpins metabolic evolvability." Mol Biol Evol 24(12);2716-22. PMID: 17884825

Rousset08: Rousset C, Fontecave M, Ollagnier de Choudens S (2008). "The [4Fe-4S] cluster of quinolinate synthase from Escherichia coli: Investigation of cluster ligands." FEBS Lett 582(19);2937-44. PMID: 18674537

Saunders08: Saunders AH, Booker SJ (2008). "Regulation of the activity of Escherichia coli quinolinate synthase by reversible disulfide-bond formation." Biochemistry 47(33);8467-9. PMID: 18651751

Suzuki73: Suzuki N, Carlson J, Griffith G, Gholson RK (1973). "Studies on the de novo biosynthesis of NAD in Escherichia coli. V. Properties of the quinolinic acid synthetase system." Biochim Biophys Acta 304(2);309-15. PMID: 4351074

Taylor67a: Taylor AL, Trotter CD (1967). "Revised linkage map of Escherichia coli." Bacteriol Rev 31(4);332-53. PMID: 4865540

Tritz70: Tritz GJ, Matney TS, Gholson RK (1970). "Mapping of the nadB locus adjacent to a previously undescribed purine locus in Escherichia coli K-12." J Bacteriol 102(2);377-81. PMID: 4315893

UniProt10: UniProt Consortium (2010). "UniProt version 2010-07 released on 2010-06-15 00:00:00." Database.

UniProt10a: UniProt Consortium (2010). "UniProt version 2010-11 released on 2010-11-02 00:00:00." Database.

UniProt11: UniProt Consortium (2011). "UniProt version 2011-06 released on 2011-06-30 00:00:00." Database.

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

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

UniProtGOA12: UniProt-GOA (2012). "Gene Ontology annotation based on UniPathway vocabulary mapping."

Other References Related to Gene Regulation

Gerasimova05: Gerasimova AV, Gelfand MS (2005). "Evolution of the NadR regulon in Enterobacteriaceae." J Bioinform Comput Biol 3(4);1007-19. PMID: 16078372

MendozaVargas09: Mendoza-Vargas A, Olvera L, Olvera M, Grande R, Vega-Alvarado L, Taboada B, Jimenez-Jacinto V, Salgado H, Juarez K, Contreras-Moreira B, Huerta AM, Collado-Vides J, Morett E (2009). "Genome-wide identification of transcription start sites, promoters and transcription factor binding sites in E. coli." PLoS One 4(10);e7526. PMID: 19838305

Tritz73: Tritz GJ, Chandler JL (1973). "Recognition of a gene involved in the regulation of nicotinamide adenine dinucleotide biosynthesis." J Bacteriol 114(1);128-36. PMID: 4349027


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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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