Escherichia coli K-12 substr. MG1655 Enzyme: NAD synthetase, NH3-dependent

Gene: nadE Accession Numbers: EG10663 (EcoCyc), b1740, ECK1738

Synonyms: efg, ntrL

Regulation Summary Diagram: ?

Regulation summary diagram for nadE

Subunit composition of NAD synthetase, NH3-dependent = [NadE]2

NAD synthetase is an essential enzyme involved in both the de novo biosynthesis and salvage of NAD+, catalyzing the final step of both pathways.

The enzyme shows a strong preference for ammonia over glutamine as the amino group donor [Imsande61] and may even have no glutamine-dependent NAD synthetase activity at all [Willison94].

The enzyme contains a P-loop-like pyrophosphatase motif [Bork94]. Crystal structures of the enzyme alone and in complex with substrates and the reaction product NAD have been solved [Jauch05].

nadE is essential for growth [Willison92, Gerdes02]. Expression of nadE is posttranscriptionally regulated by the small RNA CyaR [De09].

Locations: cytosol

Map Position: [1,820,482 -> 1,821,309] (39.24 centisomes, 141°)
Length: 828 bp / 275 aa

Molecular Weight of Polypeptide: 30.637 kD (from nucleotide sequence), 35.0 kD (experimental) [Ozment99 ]

Molecular Weight of Multimer: 60.2 kD (experimental) [Allibert87]

Unification Links: ASAP:ABE-0005803 , CGSC:28576 , DIP:DIP-10295N , EchoBASE:EB0657 , EcoGene:EG10663 , EcoliWiki:B1740 , Mint:MINT-1232772 , ModBase:P18843 , OU-Microarray:b1740 , PortEco:nadE , PR:PRO_000023335 , Pride:P18843 , Protein Model Portal:P18843 , RefSeq:NP_416254 , RegulonDB:EG10663 , SMR:P18843 , String:511145.b1740 , UniProt:P18843

Relationship Links: InterPro:IN-FAMILY:IPR003694 , InterPro:IN-FAMILY:IPR014729 , InterPro:IN-FAMILY:IPR022310 , InterPro:IN-FAMILY:IPR022926 , PDB:Structure:1WXE , PDB:Structure:1WXF , PDB:Structure:1WXG , PDB:Structure:1WXH , PDB:Structure:1WXI , Pfam:IN-FAMILY:PF02540

Gene-Reaction Schematic: ?

Gene-Reaction Schematic

Genetic Regulation Schematic: ?

Genetic regulation schematic for nadE

GO Terms:

Biological Process: GO:0006974 - cellular response to DNA damage stimulus Inferred from experiment [Khil02]
GO:0034355 - NAD salvage Inferred from experiment [Ozment99]
GO:0034628 - 'de novo' NAD biosynthetic process from aspartate Inferred from experiment [Ozment99]
GO:0009435 - NAD biosynthetic process Inferred by computational analysis [UniProtGOA12, GOA06, GOA01]
Molecular Function: GO:0008795 - NAD+ synthase activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01a, GOA01, Ozment99]
GO:0000166 - nucleotide binding Inferred by computational analysis [UniProtGOA11]
GO:0003952 - NAD+ synthase (glutamine-hydrolyzing) activity Inferred by computational analysis [GOA01]
GO:0005524 - ATP binding Inferred by computational analysis [UniProtGOA11, GOA06, GOA01]
GO:0016874 - ligase activity Inferred by computational analysis [UniProtGOA11]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08]

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

Essentiality data for nadE knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB Lennox No 37 Aerobic 7   No [Baba06, Comment 1]

Last-Curated ? 09-Jan-2009 by Keseler I , SRI International

Enzymatic reaction of: NAD+ synthetase, glutamine dependent (NAD synthetase, NH3-dependent)

Synonyms: NAD+ synthetase, glutamine-hydrolyzing, deamido-NAD+:L-glutamine amido-ligase (AMP-forming), NH(3)-dependent NAD+ synthetase

EC Number:

ATP + nicotinate adenine dinucleotide + L-glutamine + H2O <=> AMP + L-glutamate + NAD+ + diphosphate + H+

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.

The reaction is physiologically favored in the direction shown.

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

Kinetic Parameters:

Km (μM)

pH(opt): 8.5 [Imsande61]

Enzymatic reaction of: NAD+ synthetase, NH3-dependent (NAD synthetase, NH3-dependent)

Synonyms: deamido-NAD+:ammonia ligase (AMP-forming)

EC Number:

ammonium + ATP + nicotinate adenine dinucleotide <=> AMP + NAD+ + diphosphate + H+

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.

The reaction is physiologically favored in the direction shown.

Alternative Substrates [Comment 2]:

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

Purification and characterization of the enzyme in [Spencer67] used E. coli B.

Either Mg+2 or Mn+2 could satisfy the metal ion requirement. Mn+2 was more effective at low concentrations, but at high concentrations it inhibited the reaction; Mg+2 did not [Spencer67].

Cofactors or Prosthetic Groups [Comment 3]: Mg2+ [Imsande61, Ozment99]

Alternative Cofactors for Mg2+: Mn2+

Inhibitors (Unknown Mechanism): ADP [Spencer67, Comment 4] , AMP [Spencer67, Comment 4] , 3'-deoxyadenosine [Spencer67, Comment 4] , adenosine [Spencer67] , decoyinine [Spencer67] , psicofuranine [Spencer67, Comment 5]

Kinetic Parameters:

Km (μM)
nicotinate adenine dinucleotide

pH(opt): 8-8.5 [Ozment99]

Sequence Features

Protein sequence of NadE with features indicated

Feature Class Location Attached Group Citations Comment
Sequence-Conflict 13 -> 31  
[Allibert87, UniProt10a]
UniProt: (in Ref. 1; AAA79852);
Amino-Acid-Sites-That-Bind 33  
UniProt: NAD.
Nucleotide-Phosphate-Binding-Region 46 -> 53 ATP
UniProt: ATP.
Amino-Acid-Sites-That-Bind 82  
UniProt: ATP.
Amino-Acid-Sites-That-Bind 88  
UniProt: ATP; Non-Experimental Qualifier: by similarity;
Amino-Acid-Sites-That-Bind 140  
UniProt: NAD.
Amino-Acid-Sites-That-Bind 160  
UniProt: ATP.
Nucleotide-Phosphate-Binding-Region 170 -> 180 NAD+
UniProt: NAD.
Amino-Acid-Sites-That-Bind 211  
UniProt: ATP; Non-Experimental Qualifier: by similarity;
Amino-Acid-Sites-That-Bind 261  
UniProt: NAD.

Gene Local Context (not to scale): ?

Gene local context diagram

Transcription Unit:

Transcription-unit diagram


10/20/97 Gene b1740 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10663; confirmed by SwissProt match.


Allibert87: Allibert P, Willison JC, Vignais PM (1987). "Complementation of nitrogen-regulatory (ntr-like) mutations in Rhodobacter capsulatus by an Escherichia coli gene: cloning and sequencing of the gene and characterization of the gene product." J Bacteriol 169(1);260-71. PMID: 3025172

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

Bork94: Bork P, Koonin EV (1994). "A P-loop-like motif in a widespread ATP pyrophosphatase domain: implications for the evolution of sequence motifs and enzyme activity." Proteins 20(4);347-55. PMID: 7731953

De09: De Lay N, Gottesman S (2009). "The Crp-activated small noncoding regulatory RNA CyaR (RyeE) links nutritional status to group behavior." J Bacteriol 191(2);461-76. PMID: 18978044

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

Gerdes02: Gerdes SY, Scholle MD, D'Souza M, Bernal A, Baev MV, Farrell M, Kurnasov OV, Daugherty MD, Mseeh F, Polanuyer BM, Campbell JW, Anantha S, Shatalin KY, Chowdhury SA, Fonstein MY, Osterman AL (2002). "From genetic footprinting to antimicrobial drug targets: examples in cofactor biosynthetic pathways." J Bacteriol 184(16);4555-72. PMID: 12142426

GOA01: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

GOA01a: GOA, MGI (2001). "Gene Ontology annotation based on Enzyme Commission mapping." Genomics 74;121-128.

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

Imsande61: Imsande J (1961). "Pathway of diphosphopyridine nucleotide biosynthesis in Escherichia coli." J Biol Chem 1961;236(5):1494-1497. PMID: 13717628

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

Jauch05: Jauch R, Humm A, Huber R, Wahl MC (2005). "Structures of Escherichia coli NAD synthetase with substrates and products reveal mechanistic rearrangements." J Biol Chem 280(15);15131-40. PMID: 15699042

Khil02: Khil PP, Camerini-Otero RD (2002). "Over 1000 genes are involved in the DNA damage response of Escherichia coli." Mol Microbiol 44(1);89-105. PMID: 11967071

Ozment99: Ozment C, Barchue J, DeLucas LJ, Chattopadhyay D (1999). "Structural study of Escherichia coli NAD synthetase: overexpression, purification, crystallization, and preliminary crystallographic analysis." J Struct Biol 127(3);279-82. PMID: 10544053

Spencer67: Spencer RL, Preiss J (1967). "Biosynthesis of diphosphopyridine nucleotide. The purification and the properties of diphospyridine nucleotide synthetase from Escherichia coli b." J Biol Chem 1967;242(3);385-92. PMID: 4290215

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.

UniProt15: UniProt Consortium (2015). "UniProt version 2015-01 released on 2015-01-16 00:00:00." Database.

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

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

Willison92: Willison JC (1992). "An essential gene (efg) located at 38.1 minutes on the Escherichia coli chromosome." J Bacteriol 174(17);5765-6. PMID: 1512214

Willison94: Willison JC, Tissot G (1994). "The Escherichia coli efg gene and the Rhodobacter capsulatus adgA gene code for NH3-dependent NAD synthetase." J Bacteriol 1994;176(11);3400-2. PMID: 8195100

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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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