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Escherichia coli K-12 substr. MG1655 Enzyme: N5-carboxyaminoimidazole ribonucleotide synthetase



Gene: purK Accession Numbers: EG10796 (EcoCyc), b0522, ECK0515

Synonyms: purE2, purE2 peptide, CO2-binding subunit, CO2 subunit, AIR carboxylase

Regulation Summary Diagram: ?

Subunit composition of N5-carboxyaminoimidazole ribonucleotide synthetase = [PurK]2
         N5-carboxyaminoimidazole ribonucleotide synthetase monomer = PurK

Summary:
PurK and PurE were previously thought to be two subunits of AIR carboxylase [Tiedeman89], although later studies showed the enzymes to be subunits of a distinct carboxylase and mutase, respectively [Mueller94a].

PurK converts 5-amino-1-(5-phospho-D-ribosyl)imidazole (AIR) to the unstable intermediate N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) (the carbamate of AIR) in an ATP-dependent manner by the ligation of bicarbonate to the N5 amino group of AIR [Mueller94a, Zhang08f].

Crystal structures have been determined for PurK in complex with sulfate and MgADP [Thoden99]. A later report refined the crystal structure for PurK to 1.6 Å resolution with MgATP, or MgADP/P(i) bound to the active site. The proposed reaction mechanism involves a carboxyphosphate intermediate. The enzyme is not found in humans and is of interest as a target for the design of antimicrobial drugs [Thoden08]. While this three domain protein is a member of the ATP grasp superfamily, it lacks conservation within the substrate specificity (Ω) loop [Thoden99] .

PurK exhibits AIR-dependent ATPase activity that does not show bicarbonate dependence and AIR is not carboxylated during ATP hydrolysis [Meyer92]. A high concentration of bicarbonate partially rescues the defect of a purK mutant during growth in the absence of purines, probably by perturbing the balance of AIR toward N5-CAIR. An overproduction of PurE further increases rescue in the presence of bicarbonate [Firestine94]. Nonenzymatic carboxylation of AIR occurs, although under physiological conditions ATP must be added as a second substrate for the AIR carboxylation reaction to occur. PurK is required for AIR conversion to 5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxylate (CAIR) under low bicarbonate concentrations [Meyer92].

The overproduction and purification of PurK has been reported [Meyer92].

Analysis of the purE locus at the nucleotide sequence level revealed that the purE1 and purE2 cistrons correspond to two distinct, overlapping genes, purE and purK [Watanabe89].

In the area of enzyme engineering, domain exchange studies between the structurally homologous E. coli PurK and PurT revealed that domain-swapped hybrids could catalyze partial reactions, but further modifications were necessary for catalysis of the full reaction. These studies were consistent with the proposal of domain swapping as a mechanism for protein evolution [Li09c].

Review: [Zhang08f]

Gene Citations: [Koduri80, Meng90]

Locations: cytosol

Map Position: [550,750 <- 551,817] (11.87 centisomes)
Length: 1068 bp / 355 aa

Molecular Weight of Polypeptide: 39.461 kD (from nucleotide sequence), 39.0 kD (experimental) [Meyer92 ]

Molecular Weight of Multimer: 79.0 kD (experimental) [Meyer92]

Unification Links: ASAP:ABE-0001794 , CGSC:17995 , EchoBASE:EB0789 , EcoGene:EG10796 , EcoliWiki:b0522 , ModBase:P09029 , OU-Microarray:b0522 , PortEco:purK , PR:PRO_000023641 , Pride:P09029 , Protein Model Portal:P09029 , RefSeq:NP_415055 , RegulonDB:EG10796 , SMR:P09029 , String:511145.b0522 , UniProt:P09029

Relationship Links: InterPro:IN-FAMILY:IPR003135 , InterPro:IN-FAMILY:IPR005875 , InterPro:IN-FAMILY:IPR011054 , InterPro:IN-FAMILY:IPR011761 , InterPro:IN-FAMILY:IPR013816 , InterPro:IN-FAMILY:IPR016185 , PDB:Structure:1B6R , PDB:Structure:1B6S , PDB:Structure:3ETH , PDB:Structure:3ETJ , Pfam:IN-FAMILY:PF02222 , Prosite:IN-FAMILY:PS50975

In Paralogous Gene Group: 149 (2 members)

Gene-Reaction Schematic: ?

Genetic Regulation Schematic: ?

GO Terms:

Biological Process: GO:0006164 - purine nucleotide biosynthetic process Inferred by computational analysis [UniProtGOA11a]
GO:0006189 - 'de novo' IMP biosynthetic process Inferred by computational analysis [UniProtGOA12, GOA01]
Molecular Function: GO:0004638 - phosphoribosylaminoimidazole carboxylase activity Inferred from experiment Inferred by computational analysis [GOA01, Mueller94a]
GO:0005524 - ATP binding Inferred from experiment Inferred by computational analysis [UniProtGOA11a, GOA01, Mueller94a, Thoden08]
GO:0034028 - 5-(carboxyamino)imidazole ribonucleotide synthase activity Inferred from experiment Inferred by computational analysis [GOA01a, Mueller94a]
GO:0042803 - protein homodimerization activity Inferred from experiment [Meyer92]
GO:0000166 - nucleotide binding Inferred by computational analysis [UniProtGOA11a]
GO:0016874 - ligase activity Inferred by computational analysis [UniProtGOA11a]
GO:0046872 - metal ion binding Inferred by computational analysis [GOA01]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08]
GO:0005737 - cytoplasm

MultiFun Terms: metabolism biosynthesis of building blocks nucleotides purine biosynthesis

Essentiality data for purK 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:
Last-Curated ? 15-Dec-2011 by Fulcher C , SRI International


Enzymatic reaction of: N5-carboxyaminoimidazole ribonucleotide synthetase

Synonyms: N5-carboxyaminoimidazole ribonucleotide synthase, N5-CAIR synthase, N5-CAIR synthetase, 5-(carboxyamino)imidazole ribonucleotide synthetase, 5-(carboxyamino)imidazole ribonucleotide synthase, N5-AIR carboxylase

EC Number: 6.3.4.18

5-amino-1-(5-phospho-β-D-ribosyl)imidazole + ATP + hydrogen carbonate <=> N5-carboxyaminoimidazole ribonucleotide + ADP + phosphate + 2 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 histidine, purine, and pyrimidine biosynthesis , superpathway of purine nucleotides de novo biosynthesis II , inosine-5'-phosphate biosynthesis I

Summary:
PurK catalyzed production of N5-carboxyaminoimidazole ribonucleotide, ADP, and phosphate from 5-amino-1-(5-phospho-D-ribosyl)imidazole, bicarbonate and ATP was monitored using a pyruvate kinase/lactate dehydrogenase coupled assay in which NADH consumption was followed spectrophotometrically [Mueller94a].

Kinetic Parameters:

Substrate
Km (μM)
kcat (sec-1)
kcat/Km (sec-1 μM-1)
Citations
5-amino-1-(5-phospho-β-D-ribosyl)imidazole
66.0
[Li09c, BRENDA14]
ATP
48.0
5.9
[Li09c, BRENDA14]


Sequence Features

Feature Class Location Citations Comment
Sequence-Conflict 64 -> 65
[Tiedeman89, UniProt10]
Alternate sequence: RH → PD; UniProt: (in Ref. 1; CAA31421);
Amino-Acid-Sites-That-Bind 80
[UniProt10]
UniProt: ATP;
Conserved-Region 84 -> 267
[UniProt09]
UniProt: ATP-grasp;
Amino-Acid-Sites-That-Bind 120
[UniProt10]
UniProt: ATP;
Amino-Acid-Sites-That-Bind 131
[UniProt10]
UniProt: ATP;
Nucleotide-Phosphate-Binding-Region 153 -> 156
[UniProt10]
UniProt: ATP;
Amino-Acid-Sites-That-Bind 161
[UniProt10]
UniProt: ATP;
Nucleotide-Phosphate-Binding-Region 237 -> 238
[UniProt10]
UniProt: ATP;


Gene Local Context (not to scale): ?

Transcription Unit:

Notes:

History:
10/20/97 Gene b0522 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10796; 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

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

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

Firestine94: Firestine SM, Poon SW, Mueller EJ, Stubbe J, Davisson VJ (1994). "Reactions catalyzed by 5-aminoimidazole ribonucleotide carboxylases from Escherichia coli and Gallus gallus: a case for divergent catalytic mechanisms." Biochemistry 1994;33(39);11927-34. PMID: 7918411

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.

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

Koduri80: Koduri RK, Gots JS (1980). "A DNA-binding protein with specificity for pur genes in Escherichia coli." J Biol Chem 1980;255(20);9594-8. PMID: 7000760

Li09c: Li H, Fast W, Benkovic SJ (2009). "Structural and functional modularity of proteins in the de novo purine biosynthetic pathway." Protein Sci 18(5);881-92. PMID: 19384989

Meng90: Meng LM, Kilstrup M, Nygaard P (1990). "Autoregulation of PurR repressor synthesis and involvement of purR in the regulation of purB, purC, purL, purMN and guaBA expression in Escherichia coli." Eur J Biochem 1990;187(2);373-9. PMID: 2404765

Meyer92: Meyer E, Leonard NJ, Bhat B, Stubbe J, Smith JM (1992). "Purification and characterization of the purE, purK, and purC gene products: identification of a previously unrecognized energy requirement in the purine biosynthetic pathway." Biochemistry 1992;31(21);5022-32. PMID: 1534690

Mueller94a: Mueller EJ, Meyer E, Rudolph J, Davisson VJ, Stubbe J (1994). "N5-carboxyaminoimidazole ribonucleotide: evidence for a new intermediate and two new enzymatic activities in the de novo purine biosynthetic pathway of Escherichia coli." Biochemistry 1994;33(8);2269-78. PMID: 8117684

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

Thoden08: Thoden JB, Holden HM, Firestine SM (2008). "Structural analysis of the active site geometry of N5-carboxyaminoimidazole ribonucleotide synthetase from Escherichia coli." Biochemistry 47(50);13346-53. PMID: 19053251

Thoden99: Thoden JB, Kappock TJ, Stubbe J, Holden HM (1999). "Three-dimensional structure of N5-carboxyaminoimidazole ribonucleotide synthetase: a member of the ATP grasp protein superfamily." Biochemistry 1999;38(47);15480-92. PMID: 10569930

Tiedeman89: Tiedeman AA, Keyhani J, Kamholz J, Daum HA, Gots JS, Smith JM (1989). "Nucleotide sequence analysis of the purEK operon encoding 5'-phosphoribosyl-5-aminoimidazole carboxylase of Escherichia coli K-12." J Bacteriol 1989;171(1);205-12. PMID: 2464576

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

UniProt10: UniProt Consortium (2010). "UniProt version 2010-11 released on 2010-11-02 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."

Watanabe89: Watanabe W, Sampei G, Aiba A, Mizobuchi K (1989). "Identification and sequence analysis of Escherichia coli purE and purK genes encoding 5'-phosphoribosyl-5-amino-4-imidazole carboxylase for de novo purine biosynthesis." J Bacteriol 1989;171(1);198-204. PMID: 2644189

Zhang08f: Zhang Y, Morar M, Ealick SE (2008). "Structural biology of the purine biosynthetic pathway." Cell Mol Life Sci 65(23);3699-724. PMID: 18712276

Other References Related to Gene Regulation

Cho11: Cho BK, Federowicz SA, Embree M, Park YS, Kim D, Palsson BO (2011). "The PurR regulon in Escherichia coli K-12 MG1655." Nucleic Acids Res 39(15);6456-64. PMID: 21572102

He90a: He B, Shiau A, Choi KY, Zalkin H, Smith JM (1990). "Genes of the Escherichia coli pur regulon are negatively controlled by a repressor-operator interaction." J Bacteriol 1990;172(8);4555-62. PMID: 2198266

Mitchell03: Mitchell JE, Zheng D, Busby SJ, Minchin SD (2003). "Identification and analysis of 'extended -10' promoters in Escherichia coli." Nucleic Acids Res 31(16);4689-95. PMID: 12907708


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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 Thu Dec 18, 2014, biocyc13.