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Escherichia coli K-12 substr. MG1655 Enzyme: asparaginase I



Gene: ansA Accession Numbers: EG10045 (EcoCyc), b1767, ECK1765

Synonyms: AnsI

Regulation Summary Diagram: ?

Subunit composition of asparaginase I = [AnsA]4

Summary:
E. coli encodes three enzymes with asparaginase activity [Schwartz66, Cedar67, Borek00]. Asparaginase I (AnsA) is cytoplasmic and has a comparatively low affinity for its substrate, but allows growth on L-asparagine as the sole source of nitrogen [Willis74, Del83]. Unlike asparaginase II, it has no therapeutic activity as a treatment for acute lymphoblastic leukemia.

Crystal structures of AnsA have been solved [Yun07]. Although AnsA was initially thought to form a dimer in solution [Jerlstrom89], it was shown to be a tetramer arranged as a dimer of dimers. The co-crystal structure in the presence of L-asparagine revealed the presence of an allosteric binding site. A variety of site-directed mutants were generated based on the crystal structure, and their enzymatic acitivities were assayed [Yun07].

An ansA mutant is unable to utilize L-asparagine as the sole source of nitrogen [Del83].

AnsA: "asparaginase" [Del83]

Locations: cytosol

Map Position: [1,848,884 -> 1,849,900] (39.85 centisomes)
Length: 1017 bp / 338 aa

Molecular Weight of Polypeptide: 37.127 kD (from nucleotide sequence), 43.0 kD (experimental) [Spring86 ]

Isozyme Sequence Similarity [Comment 1]:

Unification Links: ASAP:ABE-0005886 , CGSC:1030 , EchoBASE:EB0043 , EcoGene:EG10045 , EcoliWiki:b1767 , ModBase:P0A962 , OU-Microarray:b1767 , PortEco:ansA , PR:PRO_000022103 , Pride:P0A962 , Protein Model Portal:P0A962 , RefSeq:NP_416281 , RegulonDB:EG10045 , SMR:P0A962 , String:511145.b1767 , UniProt:P0A962

Relationship Links: InterPro:IN-FAMILY:IPR006033 , InterPro:IN-FAMILY:IPR006034 , InterPro:IN-FAMILY:IPR020827 , InterPro:IN-FAMILY:IPR027473 , InterPro:IN-FAMILY:IPR027474 , InterPro:IN-FAMILY:IPR027475 , PDB:Structure:2HIM , PDB:Structure:2P2D , PDB:Structure:2P2N , Pfam:IN-FAMILY:PF00710 , Prints:IN-FAMILY:PR00139 , Prosite:IN-FAMILY:PS00144 , Prosite:IN-FAMILY:PS00917 , Smart:IN-FAMILY:SM00870

In Paralogous Gene Group: 473 (2 members)

Gene-Reaction Schematic: ?

GO Terms:

Biological Process: GO:0033345 - asparagine catabolic process via L-aspartate Inferred from experiment [Willis74, Del83]
GO:0006520 - cellular amino acid metabolic process Inferred by computational analysis [GOA01]
GO:0008152 - metabolic process Inferred by computational analysis [UniProtGOA11a]
Molecular Function: GO:0004067 - asparaginase activity Inferred from experiment Inferred by computational analysis [GOA01a, GOA01, Yun07]
GO:0042802 - identical protein binding Inferred from experiment [Yun07]
GO:0003824 - catalytic activity Inferred by computational analysis [UniProtGOA11a]
GO:0016787 - hydrolase activity Inferred by computational analysis [UniProtGOA11a]
Cellular Component: GO:0005737 - cytoplasm Inferred from experiment Inferred by computational analysis [UniProtGOA11, UniProtGOA11a, Cedar67]
GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, LopezCampistrou05, Cedar67]

MultiFun Terms: metabolism carbon utilization amino acids

Essentiality data for ansA knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB enriched Yes 37 Aerobic 6.95   Yes [Gerdes03, Comment 2]
LB Lennox Yes 37 Aerobic 7   Yes [Baba06, Comment 3]
M9 medium with 1% glycerol Yes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 4]
MOPS medium with 0.4% glucose Yes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 3]
Yes [Feist07, Comment 5]

Credits:
Last-Curated ? 19-Oct-2011 by Keseler I , SRI International


Enzymatic reaction of: asparaginase

Synonyms: L-asparagine aminohydrolase, L-asparaginase

EC Number: 3.5.1.1

L-asparagine + H2O <=> L-aspartate + ammonium

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: asparagine degradation I , superpathway of aspartate and asparagine biosynthesis; interconversion of aspartate and asparagine

Summary:
The enzyme exhibits strong positive cooperativity with a Hill coefficient of 2.6. Half maximal velocity is achieved at 1.2 mM L-asparagine [Yun07].

Activators (Allosteric): L-asparagine [Yun07]

Inhibitors (Competitive): 5-diazo-4-oxo-norvaline (Kic = 2600µM) [Willis74]

Primary Physiological Regulators of Enzyme Activity: L-asparagine

Kinetic Parameters:

Substrate
Km (μM)
kcat (sec-1)
kcat/Km (sec-1 μM-1)
Citations
L-asparagine
11.5
[Wriston73, BRENDA14]
L-asparagine
12.5
[Wriston85, BRENDA14]
L-asparagine
442.0
[Qiao10, BRENDA14]
L-asparagine
3500.0
[Willis74]
L-asparagine
15.0
24.0
[Derst00, BRENDA14]

T(opt): 50 °C [BRENDA14, Zhang04], 60 °C [BRENDA14, Zhang04]

pH(opt): 8 [BRENDA14, Wriston85]


Sequence Features

Feature Class Location Citations Comment
Mutagenesis-Variant 14
[Yun07, UniProt11]
Alternate sequence: T → V; UniProt: Loss of enzyme activity.
Alternate sequence: T → A; UniProt: Loss of enzyme activity.
Active-Site 14
[Yun07, UniProt11]
UniProt: O-isoaspartyl threonine intermediate.
Protein-Segment 59 -> 61
[UniProt10a]
UniProt: Substrate binding; Sequence Annotation Type: region of interest;
Mutagenesis-Variant 61
[UniProt10a]
Alternate sequence: S → Q; UniProt: Loss of enzyme activity;
Mutagenesis-Variant 91
[Yun07, UniProt11]
Alternate sequence: T → V; UniProt: Loss of enzyme activity.
Alternate sequence: T → A; UniProt: Loss of enzyme activity.
Protein-Segment 91 -> 92
[UniProt10a]
UniProt: Substrate binding; Sequence Annotation Type: region of interest;
Mutagenesis-Variant 118
[Yun07, UniProt11]
Alternate sequence: Q → D; UniProt: Loss of enzyme activity.
Mutagenesis-Variant 162
[Yun07, UniProt11]
Alternate sequence: T → A; UniProt: No effect on activity at saturating substrate concentration. Abolishes cooperativity.
Amino-Acid-Sites-That-Bind 162
[UniProt10a]
UniProt: Allosteric activator;
Mutagenesis-Variant 240
[Yun07, UniProt11]
Alternate sequence: R → A; UniProt: No effect on activity at saturating substrate concentration. Reduced activity at lower substrate concentrations.
Amino-Acid-Sites-That-Bind 240
[UniProt10a]
UniProt: Allosteric activator;
Protein-Segment 271 -> 273
[UniProt10a]
UniProt: Allosteric activator binding; Sequence Annotation Type: region of interest;


Gene Local Context (not to scale): ?

Transcription Unit:

Notes:

History:
10/20/97 Gene b1767 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10045; 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

Borek00: Borek D, Jaskolski M (2000). "Crystallization and preliminary crystallographic studies of a new L-asparaginase encoded by the Escherichia coli genome." Acta Crystallogr D Biol Crystallogr 2000;56 ( Pt 11);1505-7. PMID: 11053866

BRENDA14: BRENDA team (2014). "Imported from BRENDA version existing on Aug 2014." http://www.brenda-enzymes.org.

Cedar67: Cedar H, Schwartz JH (1967). "Localization of the two-L-asparaginases in anaerobically grown Escherichia coli." J Biol Chem 242(16);3753-5. PMID: 4962587

Del83: Del Casale T, Sollitti P, Chesney RH (1983). "Cytoplasmic L-asparaginase: isolation of a defective strain and mapping of ansA." J Bacteriol 154(1);513-5. PMID: 6339481

Derst00: Derst C, Henseling J, Rohm KH (2000). "Engineering the substrate specificity of Escherichia coli asparaginase. II. Selective reduction of glutaminase activity by amino acid replacements at position 248." Protein Sci 9(10);2009-17. PMID: 11106175

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

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

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."

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

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

Jerlstrom89: Jerlstrom PG, Bezjak DA, Jennings MP, Beacham IR (1989). "Structure and expression in Escherichia coli K-12 of the L-asparaginase I-encoding ansA gene and its flanking regions." Gene 1989;78(1);37-46. PMID: 2670682

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

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

Qiao10: Qiao J, Qi L, Ma H, Chen Y, Wang M, Wang D (2010). "Study on amino amides and enzyme kinetics of L-asparaginase by MCE." Electrophoresis 31(9);1565-71. PMID: 20358537

Schwartz66: Schwartz JH, Reeves JY, Broome JD (1966). "Two L-asparaginases from E. coli and their action against tumors." Proc Natl Acad Sci U S A 56(5);1516-9. PMID: 5339624

Spring86: Spring KJ, Jerlstrom PG, Burns DM, Beacham IR (1986). "L-asparaginase genes in Escherichia coli: isolation of mutants and characterization of the ansA gene and its protein product." J Bacteriol 1986;166(1);135-42. PMID: 3514575

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 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."

Willis74: Willis RC, Woolfolk CA (1974). "Asparagine utilization in Escherichia coli." J Bacteriol 118(1);231-41. PMID: 4595199

Wriston73: Wriston JC, Yellin TO (1973). "L-asparaginase: a review." Adv Enzymol Relat Areas Mol Biol 39;185-248. PMID: 4583638

Wriston85: Wriston JC (1985). "Asparaginase." Methods Enzymol 113;608-18. PMID: 3911011

Yun07: Yun MK, Nourse A, White SW, Rock CO, Heath RJ (2007). "Crystal structure and allosteric regulation of the cytoplasmic Escherichia coli L-asparaginase I." J Mol Biol 369(3);794-811. PMID: 17451745

Zhang04: Zhang YQ, Tao ML, Shen WD, Zhou YZ, Ding Y, Ma Y, Zhou WL (2004). "Immobilization of L-asparaginase on the microparticles of the natural silk sericin protein and its characters." Biomaterials 25(17);3751-9. PMID: 15020151


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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 18.5 on Fri Dec 19, 2014, BIOCYC13A.