|Gene:||purK||Accession Numbers: EG10796 (MetaCyc), b0522, ECK0515|
Synonyms: purE2, purE2 peptide, CO2-binding subunit, CO2 subunit, AIR carboxylase
Species: Escherichia coli K-12 substr. MG1655
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, Zhang08c].
Crystal structures have been determined for PurK in complex with sulfate and MgADP [Thoden99b]. 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 [Thoden99b] .
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 [Li09a].
|Map Position: [550,750 <- 551,817]|
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
|Biological Process:||GO:0006164 - purine nucleotide biosynthetic process
GO:0006189 - 'de novo' IMP biosynthetic process [UniProtGOA12, GOA01a]
|Molecular Function:||GO:0004638 - phosphoribosylaminoimidazole carboxylase activity
GO:0005524 - ATP binding [UniProtGOA11a, GOA01a, Mueller94a, Thoden08]
GO:0034028 - 5-(carboxyamino)imidazole ribonucleotide synthase activity [GOA01, Mueller94a]
GO:0042803 - protein homodimerization activity [Meyer92]
GO:0000166 - nucleotide binding [UniProtGOA11a]
GO:0016874 - ligase activity [UniProtGOA11a]
GO:0046872 - metal ion binding [GOA01a]
|Cellular Component:||GO:0005829 - cytosol
GO:0005737 - cytoplasm
|MultiFun Terms:||metabolism → biosynthesis of building blocks → nucleotides → purine biosynthesis|
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 Enzyme Commission system.
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 [Mueller94a].
|Sequence-Conflict||64 -> 65|
|Conserved-Region||84 -> 267|
|Nucleotide-Phosphate-Binding-Region||153 -> 156|
|Nucleotide-Phosphate-Binding-Region||237 -> 238|
10/20/97 Gene b0522 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10796; confirmed by SwissProt match.
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
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
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
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
Thoden99b: 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
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