|Gene:||purK||Accession Numbers: EG10796 (EcoCyc), b0522, ECK0515|
Synonyms: purE2, purE2 peptide, CO2-binding subunit, CO2 subunit, AIR carboxylase
Subunit composition of
N5-carboxyaminoimidazole ribonucleotide synthetase = [PurK]2
N5-carboxyaminoimidazole ribonucleotide synthetase monomer = PurK
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 [Mueller94].
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 [Mueller94, Zhang08].
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 [Li09].
|Map Position: [550,750 <- 551,817] (11.87 centisomes, 43°)||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)
|Biological Process:||GO:0006164 - purine nucleotide biosynthetic process
GO:0006189 - 'de novo' IMP biosynthetic process [UniProtGOA12, GOA06, GOA01]
|Molecular Function:||GO:0004638 - phosphoribosylaminoimidazole carboxylase activity
GO:0005524 - ATP binding [UniProtGOA11, GOA01, Mueller94, Thoden08]
GO:0034028 - 5-(carboxyamino)imidazole ribonucleotide synthase activity [GOA06, GOA01a, Mueller94]
GO:0042803 - protein homodimerization activity [Meyer92]
GO:0000166 - nucleotide binding [UniProtGOA11]
GO:0016874 - ligase activity [UniProtGOA11]
GO:0046872 - metal ion binding [GOA01]
|Cellular Component:||GO:0005829 - cytosol
GO:0005737 - cytoplasm
|MultiFun Terms:||metabolism → biosynthesis of building blocks → nucleotides → purine biosynthesis|
|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]
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: 126.96.36.199
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.
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 [Mueller94].
|Feature Class||Location||Attached Group||Citations||Comment|
|Sequence-Conflict||64 -> 65|
|Conserved-Region||84 -> 267|
|Nucleotide-Phosphate-Binding-Region||125 -> 131||ATP|
|Nucleotide-Phosphate-Binding-Region||153 -> 156||ATP|
|Nucleotide-Phosphate-Binding-Region||237 -> 238||ATP|
10/20/97 Gene b0522 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10796; confirmed by SwissProt match.
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
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
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
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
Mueller94: 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
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
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
He90: 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
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