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Escherichia coli K-12 substr. MG1655 Enzyme: phosphoribosylglycinamide formyltransferase 2



Gene: purT Accession Numbers: EG11809 (EcoCyc), b1849, ECK1850

Synonyms: GART2, glycinamide ribonucleotide transformylase 2, 5'-phosphoribosylglycinamide transformylase 2, GAR transformylase 2, GAR transformylase T

Regulation Summary Diagram: ?

Summary:
E. coli contains two different phosphoribosylglycinamide (GAR) transformylases, both of which can catalyze the third step in de novo purine biosynthesis. The transformylase encoded by purN utilizes 10-formyl-tetrahydrofolate as the formyl donor. The second transformylase encoded by purT utilizes formate, which is provided by PurU-catalyzed hydrolysis of 10-formyl-tetrahydrofolate [Nagy93]. The existence of these two transformylase enzymes was determined by mutant studies. A strain containing a knockout insertion in purN did not result in purine auxotrophy [Smith87]. Only mutants defective in both purN and purT required exogenously added purine for growth [Nygaard93]. There is no significant homology between the two transformylases [Marolewski94].

Kinetic studies of wild-type and mutant PurT, as well as positional isotope exchange studies, demonstrated a formyl phosphate intermediate in the catalytic mechanism of the GAR transformylase reaction [Marolewski97]. GAR transformylase 2 can also catalyze a second, side reaction that is different from its transformylase activity. The second activity is acetate kinase, which cleaves ATP in the presence of acetate to produce acetylphosphate and ADP [Marolewski94].

The crystal structure of phosphoribosylglycinamide transformylase 2 has been determined in complex with substrate and/or nucleotide analogs [Thoden00, Thoden02]. The enzyme was determined to be a monomer in solution [Marolewski94], but a homodimer in the crystal [Thoden00]. It belongs to the ATP-grasp superfamily [Thoden00].

Review: Jensen, K.F., G. Dandanell, B. Hove-Jensen, and M. Willemoes (2008) "Nucleotides, Nucleosides and Nucleobases" EcoSal 3.6.2 [EcoSal]

Locations: cytosol

Map Position: [1,928,905 -> 1,930,083] (41.57 centisomes)
Length: 1179 bp / 392 aa

Molecular Weight of Polypeptide: 42.434 kD (from nucleotide sequence), 41.9 kD (experimental) [Marolewski94 ]

pI: 5.74

Isozyme Sequence Similarity:
phosphoribosylglycinamide formyltransferase 1: NO

Unification Links: ASAP:ABE-0006162 , CGSC:32348 , DIP:DIP-10618N , EchoBASE:EB1757 , EcoGene:EG11809 , EcoliWiki:b1849 , Mint:MINT-1306001 , ModBase:P33221 , OU-Microarray:b1849 , PortEco:purT , PR:PRO_000023645 , Pride:P33221 , Protein Model Portal:P33221 , RefSeq:NP_416363 , RegulonDB:EG11809 , SMR:P33221 , String:511145.b1849 , UniProt:P33221

Relationship Links: InterPro:IN-FAMILY:IPR003135 , InterPro:IN-FAMILY:IPR005862 , InterPro:IN-FAMILY:IPR011054 , InterPro:IN-FAMILY:IPR011761 , InterPro:IN-FAMILY:IPR013815 , InterPro:IN-FAMILY:IPR013816 , InterPro:IN-FAMILY:IPR016185 , Panther:IN-FAMILY:PTHR23047:SF2 , PDB:Structure:1EYZ , PDB:Structure:1EZ1 , PDB:Structure:1KJ8 , PDB:Structure:1KJ9 , PDB:Structure:1KJI , PDB:Structure:1KJJ , PDB:Structure:1KJQ , PDB:Structure:1NFE , Pfam:IN-FAMILY:PF02222 , Prosite:IN-FAMILY:PS50975

In Paralogous Gene Group: 149 (2 members)

Gene-Reaction Schematic: ?

GO Terms:

Biological Process: GO:0016310 - phosphorylation Inferred from experiment [Marolewski94]
GO:0006164 - purine nucleotide biosynthetic process Inferred by computational analysis [UniProtGOA11a]
GO:0006189 - 'de novo' IMP biosynthetic process Inferred by computational analysis [UniProtGOA12]
GO:0009152 - purine ribonucleotide biosynthetic process Inferred by computational analysis [GOA01a]
Molecular Function: GO:0005515 - protein binding Inferred from experiment [Rajagopala14, Butland05]
GO:0008776 - acetate kinase activity Inferred from experiment [Marolewski94]
GO:0000166 - nucleotide binding Inferred by computational analysis [UniProtGOA11a]
GO:0000287 - magnesium ion binding Inferred by computational analysis [GOA01a]
GO:0003824 - catalytic activity Inferred by computational analysis [GOA01a]
GO:0004644 - phosphoribosylglycinamide formyltransferase activity Inferred by computational analysis [GOA01a]
GO:0005524 - ATP binding Inferred by computational analysis [UniProtGOA11a, GOA06, GOA01a]
GO:0016740 - transferase activity Inferred by computational analysis [UniProtGOA11a]
GO:0043815 - phosphoribosylglycinamide formyltransferase 2 activity Inferred by computational analysis [GOA06]
GO:0046872 - metal ion binding Inferred by computational analysis [UniProtGOA11a, GOA01a]
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 purT 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 1% glycerol Yes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 3]
MOPS medium with 0.4% glucose Yes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 2]
Yes [Feist07, Comment 4]

Credits:
Last-Curated ? 06-Jan-2012 by Fulcher C , SRI International


Enzymatic reaction of: phosphoribosylglycinamide formyltransferase

Synonyms: GAR transformylase, 5'-phosphoribosylglycinamide transformylase, formate-dependent GAR transformylase, glycinamide ribonucleotide transformylase

EC Number: 2.1.2.-

N1-(5-phospho-β-D-ribosyl)glycinamide + formate + ATP <=> N2-formyl-N1-(5-phospho-β-D-ribosyl)glycinamide + ADP + phosphate + H+

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 favored in the direction shown.

Alternative Substrates [Comment 5]:

In Pathways: superpathway of histidine, purine, and pyrimidine biosynthesis , superpathway of purine nucleotides de novo biosynthesis II , superpathway of 5-aminoimidazole ribonucleotide biosynthesis , 5-aminoimidazole ribonucleotide biosynthesis II

Summary:
The enzyme was specific for the β-isomer of 5-phospho-ribosyl-glycineamide. It was also specific for formate and ATP as formyl donor, and NTP, respectively. The kinetic parameters shown here were determined at pH 8.0 [Marolewski94].

An equilibrium constant for the forward reaction of 2.0 +/- 0.35 x 106 at 25°C suggested low reversibility [Marolewski94].

Co2+ and Mn2+ could substitute for Mg2+ [Marolewski94].

Cofactors or Prosthetic Groups: Co2+ [Marolewski94], Mn2+ [Marolewski94], Mg2+ [Comment 6, Marolewski94]

Kinetic Parameters:

Substrate
Km (μM)
Citations
formate
319.0
[Marolewski94]
N1-(5-phospho-β-D-ribosyl)glycinamide
10.1
[Marolewski94]


Enzymatic reaction of: acetate kinase (phosphoribosylglycinamide formyltransferase 2)

EC Number: 2.7.2.1

acetate + ATP <=> acetyl phosphate + ADP

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

This reaction is reversible.

Summary:
The kinetic parameters shown here were determined at pH 8.0 [Marolewski94].

Kinetic Parameters:

Substrate
Km (μM)
Citations
acetate
3680.0
[Marolewski94]
ATP
77.4
[Marolewski94]


Sequence Features

Feature Class Location Citations Comment
Cleavage-of-Initial-Methionine 1
[Marolewski94, UniProt11a]
UniProt: Removed.
Chain 2 -> 392
[UniProt09]
UniProt: Phosphoribosylglycinamide formyltransferase 2;
Protein-Segment 22 -> 23
[UniProt10]
UniProt: 5'-phosphoribosylglycinamide binding; Sequence Annotation Type: region of interest;
Amino-Acid-Sites-That-Bind 82
[UniProt10a]
UniProt: 5'-phosphoribosylglycinamide;
Amino-Acid-Sites-That-Bind 114
[UniProt10a]
UniProt: ATP;
Conserved-Region 119 -> 308
[UniProt09]
UniProt: ATP-grasp;
Amino-Acid-Sites-That-Bind 155
[UniProt10a]
UniProt: ATP;
Nucleotide-Phosphate-Binding-Region 160 -> 165
[UniProt10a]
UniProt: ATP;
Acetylation-Modification 179
[Zhang09a, UniProt11a]
UniProt: N6-acetyllysine.
Nucleotide-Phosphate-Binding-Region 195 -> 198
[UniProt10a]
UniProt: ATP;
Amino-Acid-Sites-That-Bind 203
[UniProt10a]
UniProt: ATP;
Metal-Binding-Site 267
[UniProt10a]
UniProt: Magnesium; Non-Experimental Qualifier: by similarity;
Metal-Binding-Site 279
[UniProt10a]
UniProt: Magnesium; Non-Experimental Qualifier: by similarity;
Amino-Acid-Sites-That-Bind 286
[UniProt10a]
UniProt: 5'-phosphoribosylglycinamide;
Amino-Acid-Sites-That-Bind 355
[UniProt10a]
UniProt: 5'-phosphoribosylglycinamide;
Protein-Segment 362 -> 363
[UniProt10]
UniProt: 5'-phosphoribosylglycinamide binding; Sequence Annotation Type: region of interest;


Gene Local Context (not to scale): ?

Transcription Unit:

Notes:

History:
10/20/97 Gene b1849 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG11809; 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

Butland05: Butland G, Peregrin-Alvarez JM, Li J, Yang W, Yang X, Canadien V, Starostine A, Richards D, Beattie B, Krogan N, Davey M, Parkinson J, Greenblatt J, Emili A (2005). "Interaction network containing conserved and essential protein complexes in Escherichia coli." Nature 433(7025);531-7. PMID: 15690043

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

EcoSal: "Escherichia coli and Salmonella: Cellular and Molecular Biology." Online edition.

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

GOA01a: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

GOA06: GOA, SIB (2006). "Electronic Gene Ontology annotations created by transferring manual GO annotations between orthologous microbial proteins."

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

Marolewski94: Marolewski A, Smith JM, Benkovic SJ (1994). "Cloning and characterization of a new purine biosynthetic enzyme: a non-folate glycinamide ribonucleotide transformylase from E. coli." Biochemistry 1994;33(9);2531-7. PMID: 8117714

Marolewski97: Marolewski AE, Mattia KM, Warren MS, Benkovic SJ (1997). "Formyl phosphate: a proposed intermediate in the reaction catalyzed by Escherichia coli PurT GAR transformylase." Biochemistry 36(22);6709-16. PMID: 9184151

Nagy93: Nagy PL, McCorkle GM, Zalkin H (1993). "purU, a source of formate for purT-dependent phosphoribosyl-N-formylglycinamide synthesis." J Bacteriol 175(21);7066-73. PMID: 8226647

Nygaard93: Nygaard P, Smith JM (1993). "Evidence for a novel glycinamide ribonucleotide transformylase in Escherichia coli." J Bacteriol 1993;175(11);3591-7. PMID: 8501063

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

Smith87: Smith JM, Daum HA (1987). "Identification and nucleotide sequence of a gene encoding 5'-phosphoribosylglycinamide transformylase in Escherichia coli K12." J Biol Chem 1987;262(22);10565-9. PMID: 3301838

Thoden00: Thoden JB, Firestine S, Nixon A, Benkovic SJ, Holden HM (2000). "Molecular structure of Escherichia coli PurT-encoded glycinamide ribonucleotide transformylase." Biochemistry 39(30);8791-802. PMID: 10913290

Thoden02: Thoden JB, Firestine SM, Benkovic SJ, Holden HM (2002). "PurT-encoded glycinamide ribonucleotide transformylase. Accommodation of adenosine nucleotide analogs within the active site." J Biol Chem 277(26);23898-908. PMID: 11953435

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

UniProt10: UniProt Consortium (2010). "UniProt version 2010-07 released on 2010-06-15 00:00:00." Database.

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

UniProt11a: UniProt Consortium (2011). "UniProt version 2011-06 released on 2011-06-30 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."

Zhang09a: Zhang J, Sprung R, Pei J, Tan X, Kim S, Zhu H, Liu CF, Grishin NV, Zhao Y (2009). "Lysine acetylation is a highly abundant and evolutionarily conserved modification in Escherichia coli." Mol Cell Proteomics 8(2);215-25. PMID: 18723842


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Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
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