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Metabolic Modeling Tutorial
discounted EARLY registration ends Dec 31, 2014
BioCyc websites down
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Metabolic Modeling Tutorial
discounted EARLY registration ends Dec 31, 2014
BioCyc websites down
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MetaCyc Enzyme: asparagine synthetase B

Gene: asnB Accession Numbers: EG10092 (MetaCyc), b0674, ECK0662

Species: Escherichia coli K-12 substr. MG1655

Subunit composition of asparagine synthetase B = [AsnB]2

Summary:
Asparagine synthetase B (AsnB) is one of two asparagine synthetases in E. coli, catalyzing the glutamine-dependent and ammonia-dependent conversion of aspartate to asparagine.

AsnB is the less active of two asparagine synthetase activities in E. coli [Felton80, Humbert80, Scofield90]. Unlike related aminotransferases, AsnB lacks a histidine in its conserved glutamine aminotransferase domain [Richards92].

The amino-terminal cysteine of AsnB is important for its glutamine-dependent activity, but not for its ammonia-dependent activity [Boehlein94]. The crystal structure of a mutant with an amino-terminal alanine has been determined to 2.0 Å resolution [Larsen99].

Locations: cytosol

Map Position: [696,736 <- 698,400]

Molecular Weight of Polypeptide: 62.659 kD (from nucleotide sequence)

Unification Links: ASAP:ABE-0002292 , CGSC:994 , DIP:DIP-9177N , EchoBASE:EB0090 , EcoGene:EG10092 , EcoliWiki:b0674 , Mint:MINT-1306603 , ModBase:P22106 , OU-Microarray:b0674 , PortEco:asnB , PR:PRO_000022168 , Pride:P22106 , Protein Model Portal:P22106 , RefSeq:NP_415200 , RegulonDB:EG10092 , SMR:P22106 , String:511145.b0674 , UniProt:P22106

Relationship Links: InterPro:IN-FAMILY:IPR000583 , InterPro:IN-FAMILY:IPR001962 , InterPro:IN-FAMILY:IPR006426 , InterPro:IN-FAMILY:IPR014729 , InterPro:IN-FAMILY:IPR017932 , PDB:Structure:1CT9 , Pfam:IN-FAMILY:PF00733 , Pfam:IN-FAMILY:PF13537 , Prosite:IN-FAMILY:PS51278

Gene-Reaction Schematic: ?

GO Terms:

Biological Process: GO:0006529 - asparagine biosynthetic process Inferred from experiment Inferred by computational analysis [UniProtGOA11a, GOA01a, Boehlein94, Humbert80]
GO:0006541 - glutamine metabolic process Inferred from experiment Inferred by computational analysis [UniProtGOA11a, Humbert80]
GO:0008652 - cellular amino acid biosynthetic process Inferred from experiment Inferred by computational analysis [UniProtGOA11a, Humbert80]
GO:0009063 - cellular amino acid catabolic process Inferred from experiment [Humbert80]
GO:0008152 - metabolic process Inferred by computational analysis [GOA01a]
GO:0070981 - L-asparagine biosynthetic process Inferred by computational analysis [UniProtGOA12]
Molecular Function: GO:0004066 - asparagine synthase (glutamine-hydrolyzing) activity Inferred from experiment Inferred by computational analysis [GOA01, GOA01a, Boehlein94, Humbert80]
GO:0004071 - aspartate-ammonia ligase activity Inferred by computational analysis Inferred from experiment [Boehlein94]
GO:0005524 - ATP binding Inferred from experiment Inferred by computational analysis [UniProtGOA11a, Boehlein94, Parr96]
GO:0016597 - amino acid binding Inferred from experiment [Boehlein94, Parr96]
GO:0042802 - identical protein binding Inferred from experiment [Rajagopala14, Rajagopala12]
GO:0000166 - nucleotide binding Inferred by computational analysis [UniProtGOA11a]
GO:0016874 - ligase activity Inferred by computational analysis [UniProtGOA11a]
Cellular Component: GO:0005737 - cytoplasm Inferred from experiment [Humbert80, Cedar69]
GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08, LopezCampistrou05]

MultiFun Terms: metabolism biosynthesis of building blocks amino acids asparagine
metabolism carbon utilization amino acids

Credits:
Imported from EcoCyc 16-Sep-2014 by Paley S , SRI International


Enzymatic reaction of: L-aspartate:ammonia ligase (AMP-forming) (asparagine synthetase B)

EC Number: 6.3.1.1

L-aspartate + ammonium + ATP <=> L-asparagine + AMP + diphosphate + H+

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

The reaction is physiologically favored in the direction shown.

In Pathways: superpathway of aspartate and asparagine biosynthesis; interconversion of aspartate and asparagine , superpathway of asparagine biosynthesis , asparagine biosynthesis II

Credits:
Imported from EcoCyc 16-Sep-2014 by Paley S , SRI International

Summary:
Reported kcat values for this reaction include 0.50/s for ammonia and 0.40/s for aspartic acid [Boehlein94].

Cofactors or Prosthetic Groups: Mg2+ [Boehlein94]

Kinetic Parameters:

Substrate
Km (μM)
Citations
ammonium
17000.0
[Boehlein94]
L-aspartate
530.0
[Boehlein94]


Enzymatic reaction of: L-aspartate:L-glutamine amido-ligase (AMP-forming) (asparagine synthetase B)

EC Number: 6.3.5.4

L-glutamine + L-aspartate + ATP + H2O <=> L-glutamate + L-asparagine + AMP + diphosphate + H+

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 irreversible in the direction shown.

In Pathways: superpathway of aspartate and asparagine biosynthesis; interconversion of aspartate and asparagine , asparagine biosynthesis II , glutamine degradation I , superpathway of asparagine biosynthesis , asparagine biosynthesis I

Credits:
Imported from EcoCyc 16-Sep-2014 by Paley S , SRI International

Summary:
Reported kcat values for this reaction include 0.59/s for glutamine, 0.57/s for aspartic acid and 0.80/s for ATP [Boehlein94].

Other kinetic data reported for glutamine in this reaction include a Km of 0.12mM, a Vmax of 719nmol/mg/min and a kcat of 0.75/s [Richards92].

Cofactors or Prosthetic Groups: Mg2+ [Boehlein94]

Kinetic Parameters:

Substrate
Km (μM)
Citations
L-glutamine
660.0
[Boehlein94]
L-aspartate
850.0
[Boehlein94]
ATP
260.0
[Boehlein94]

pH(opt): 6.5-8 [Boehlein94]


Sequence Features

Feature Class Location Citations Comment
Cleavage-of-Initial-Methionine 1
[Boehlein94, UniProt13]
UniProt: Removed.
Mutagenesis-Variant 2
[Boehlein94, UniProt12]
Alternate sequence: S; UniProt: Loss of glutamine-dependent activity but no effect on ammonia- dependent asparagine synthetase activity.
Alternate sequence: A; UniProt: Loss of glutamine-dependent activity but no effect on ammonia- dependent asparagine synthetase activity.
Active-Site 2
[UniProt10a]
UniProt: For GATase activity;
Conserved-Region 2 -> 186
[UniProt09]
UniProt: Glutamine amidotransferase type-2;
Chain 2 -> 554
[UniProt09]
UniProt: Asparagine synthetase B [glutamine- hydrolyzing];
Mutagenesis-Variant 30
[Boehlein94, UniProt12]
Alternate sequence: A; UniProt: 4,5-fold decrease in glutamine affinity.
Mutagenesis-Variant 34
[Boehlein94, UniProt12]
Alternate sequence: E; UniProt: Little effect on the kinetic properties.
Alternate sequence: N; UniProt: Little effect on the kinetic properties.
Protein-Segment 50 -> 54
[UniProt12]
UniProt: Glutamine binding; Sequence Annotation Type: region of interest.
Protein-Segment 75 -> 77
[UniProt12]
UniProt: Glutamine binding; Sequence Annotation Type: region of interest.
Mutagenesis-Variant 81
[Boehlein94, UniProt12]
Alternate sequence: A; UniProt: 5-fold decrease in glutamine affinity.
Amino-Acid-Sites-That-Bind 99
[UniProt12]
UniProt: Glutamine.
Mutagenesis-Variant 105
[Boehlein94, UniProt12]
Alternate sequence: H; UniProt: Little effect on the kinetic properties.
Amino-Acid-Sites-That-Bind 233
[UniProt12]
UniProt: ATP; via carbonyl oxygen.
Amino-Acid-Sites-That-Bind 273
[UniProt12]
UniProt: ATP; via amide nitrogen and carbonyl oxygen.
Nucleotide-Phosphate-Binding-Region 347 -> 348
[UniProt12]
UniProt: ATP.
Mutagenesis-Variant 349
[Meyer10, UniProt12]
Alternate sequence: D; UniProt: 5-fold increase in affinity for asparate when assaying both the glutamine- and ammonia-dependent synthetase reactions, and 2-fold decrease in kcat for these reactions. Modifies the product glutamate/asparagine stoichiometry.
Alternate sequence: Q; UniProt: Loss of glutamine- and ammonia- dependent synthetase activity, but still exhibits glutaminase activity.
Alternate sequence: A; UniProt: Loss of glutamine- and ammonia- dependent synthetase activity, but still exhibits glutaminase activity.
Amino-Acid-Site 349
[UniProt12]
UniProt: Important for beta-aspartyl-AMP intermediate formation; Sequence Annotation Type: site.

History:
10/20/97 Gene b0674 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10092; confirmed by SwissProt match.


References

Boehlein94: Boehlein SK, Richards NG, Schuster SM (1994). "Glutamine-dependent nitrogen transfer in Escherichia coli asparagine synthetase B. Searching for the catalytic triad." J Biol Chem 269(10);7450-7. PMID: 7907328

Cedar69: Cedar H, Schwartz JH (1969). "The asparagine synthetase of Escherhic coli. I. Biosynthetic role of the enzyme, purification, and characterization of the reaction products." J Biol Chem 1969;244(15);4112-21. PMID: 4895361

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

Felton80: Felton J, Michaelis S, Wright A (1980). "Mutations in two unlinked genes are required to produce asparagine auxotrophy in Escherichia coli." J Bacteriol 142(1);221-8. PMID: 6102983

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."

Humbert80: Humbert R, Simoni RD (1980). "Genetic and biomedical studies demonstrating a second gene coding for asparagine synthetase in Escherichia coli." J Bacteriol 1980;142(1);212-20. PMID: 6102982

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

Larsen99: Larsen TM, Boehlein SK, Schuster SM, Richards NG, Thoden JB, Holden HM, Rayment I (1999). "Three-dimensional structure of Escherichia coli asparagine synthetase B: a short journey from substrate to product." Biochemistry 1999;38(49);16146-57. PMID: 10587437

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

Meyer10: Meyer ME, Gutierrez JA, Raushel FM, Richards NG (2010). "A conserved glutamate controls the commitment to acyl-adenylate formation in asparagine synthetase." Biochemistry 49(43);9391-401. PMID: 20853825

Parr96: Parr IB, Boehlein SK, Dribben AB, Schuster SM, Richards NG (1996). "Mapping the aspartic acid binding site of Escherichia coli asparagine synthetase B using substrate analogs." J Med Chem 39(12);2367-78. PMID: 8691431

Rajagopala12: Rajagopala SV, Sikorski P, Caufield JH, Tovchigrechko A, Uetz P (2012). "Studying protein complexes by the yeast two-hybrid system." Methods 58(4);392-9. PMID: 22841565

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

Richards92: Richards NG, Schuster SM (1992). "An alternative mechanism for the nitrogen transfer reaction in asparagine synthetase." FEBS Lett 1992;313(2);98-102. PMID: 1358677

Scofield90: Scofield MA, Lewis WS, Schuster SM (1990). "Nucleotide sequence of Escherichia coli asnB and deduced amino acid sequence of asparagine synthetase B." J Biol Chem 1990;265(22);12895-902. PMID: 1973930

UniProt09: UniProt Consortium (2009). "UniProt version 15.8 released on 2009-10-01 00:00:00." Database.

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

UniProt12: UniProt Consortium (2012). "UniProt version 2012-09 released on 2012-09-12 00:00:00." Database.

UniProt13: UniProt Consortium (2013). "UniProt version 2013-08 released on 2013-08-01 00:00:00." Database.

UniProtGOA11a: 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."


Report Errors or Provide Feedback
Please cite the following article in publications resulting from the use of MetaCyc: Caspi et al, Nucleic Acids Research 42:D459-D471 2014
Page generated by SRI International Pathway Tools version 18.5 on Fri Dec 19, 2014, biocyc11.