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Escherichia coli K-12 substr. MG1655 Enzyme: γ-glutamyl kinase



Gene: proB Accession Numbers: EG10768 (EcoCyc), b0242, ECK0243

Synonyms: pro(2), pro2, glutamate-5-kinase, G5K

Regulation Summary Diagram: ?

Component of: γ-glutamyl kinase-GP-reductase multienzyme complex

Subunit composition of γ-glutamyl kinase = [ProB]4

Summary:
Glutamate-5-kinase (G5K) catalyzes the first step in the synthesis of the osmoprotective amino acid proline [Smith84, PerezArellano05, PerezArellano06]. This is the primary point of control in this pathway because in E. coli proline synthesis is regulated through feedback inhibition of G5K activity [PerezArellano05]. Feedback inhibition of G5K by proline was first reported by [Baich65] using the enzyme from E. coli strain W.

E. coli G5K has an N-terminal amino acid kinase (AAK) domain and a C-terminal PUA (named after Pseudo Uridine synthases and Archaeosine-specific transglycosylases) domain. Deletion of the PUA domain of G5K has demonstrated that the AAK domain alone, like the full-length protein, can form tetramers, catalyze the formation of L-glutamate-5-phosphate and is feed-back inhibited by proline [PerezArellano05]. However, the deletion greatly diminished the Mg2+ requirement of the enzyme. It also reduced the sensitivity of the enzyme to proline mediated feedback inhibition [PerezArellano05]. A recent mutational analysis study has confirmed that the AAK domain is involved in substrate binding, catalysis, and feedback inhibition by proline [PerezArellano06].

G5K interacts with glutamate-5-semialdehyde dehydrogenase, to form the γ-glutamyl kinase-GP-reductase multienzyme complex. It has been shown that the complex formation is essential for the functioning of glutamate-5-kinase. However, it is not a requirement for the activity of glutamate-5-semialdehyde dehydrogenase [Smith84].

The recombinant E. coli G5K has been crystallized to a resolution of 2.9 Å [MarcoMarin07] and 2.5 Å [PerezArellano04, MarcoMarin07]. Further analysis of the crystal structure has revealed that E. coli G5K possess a novel "dimer of dimers" architecture and the tetramers are formed by the interaction of two dimers through their AAK domains. It further demonstrated that the tetramers are flat and elongated, and have two active centers in each face [MarcoMarin07].

Citations: [Seddon89]

Gene Citations: [Deutch84]

Locations: cytosol

Map Position: [259,612 -> 260,715] (5.6 centisomes)
Length: 1104 bp / 367 aa

Molecular Weight of Polypeptide: 39.056 kD (from nucleotide sequence)

Unification Links: ASAP:ABE-0000830 , CGSC:363 , EchoBASE:EB0761 , EcoGene:EG10768 , EcoliWiki:b0242 , ModBase:P0A7B5 , OU-Microarray:b0242 , PortEco:proB , PR:PRO_000023597 , Pride:P0A7B5 , Protein Model Portal:P0A7B5 , RefSeq:NP_414777 , RegulonDB:EG10768 , SMR:P0A7B5 , String:511145.b0242 , UniProt:P0A7B5

Relationship Links: InterPro:IN-FAMILY:IPR001048 , InterPro:IN-FAMILY:IPR001057 , InterPro:IN-FAMILY:IPR002478 , InterPro:IN-FAMILY:IPR005715 , InterPro:IN-FAMILY:IPR011529 , InterPro:IN-FAMILY:IPR015947 , InterPro:IN-FAMILY:IPR019797 , PDB:Structure:2J5T , PDB:Structure:2J5V , PDB:Structure:2W21 , Pfam:IN-FAMILY:PF00696 , Pfam:IN-FAMILY:PF01472 , Prints:IN-FAMILY:PR00474 , Prosite:IN-FAMILY:PS00902 , Prosite:IN-FAMILY:PS50890 , Smart:IN-FAMILY:SM00359

Gene-Reaction Schematic: ?

GO Terms:

Biological Process: GO:0006561 - proline biosynthetic process Inferred from experiment Inferred by computational analysis [UniProtGOA11a, GOA06, GOA01a, Csonka88]
GO:0008652 - cellular amino acid biosynthetic process Inferred by computational analysis [UniProtGOA11a]
GO:0016310 - phosphorylation Inferred by computational analysis [UniProtGOA11a]
GO:0055129 - L-proline biosynthetic process Inferred by computational analysis [UniProtGOA12]
Molecular Function: GO:0000287 - magnesium ion binding Inferred from experiment [PerezArellano05, PerezArellano06, Smith84]
GO:0004349 - glutamate 5-kinase activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01, GOA01a, PerezArellano06, PerezArellano05, Smith84]
GO:0000166 - nucleotide binding Inferred by computational analysis [UniProtGOA11a]
GO:0003723 - RNA binding Inferred by computational analysis [GOA01a]
GO:0005524 - ATP binding Inferred by computational analysis [UniProtGOA11a]
GO:0016301 - kinase activity Inferred by computational analysis [UniProtGOA11a]
GO:0016740 - transferase activity Inferred by computational analysis [UniProtGOA11a]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08]
GO:0005737 - cytoplasm Inferred by computational analysis [UniProtGOA11, UniProtGOA11a, GOA06, GOA01a]

MultiFun Terms: metabolism biosynthesis of building blocks amino acids proline

Essentiality data for proB 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 No 37 Aerobic 7.2 0.22 No [Baba06, Comment 2]
No [Feist07, Comment 4]

Enzymatic reaction of: γ-glutamyl kinase

Synonyms: glutamate 5-kinase, GK, ATP:L-glutamate 5-phosphotransferase, G5K, gamma-glutamate kinase

EC Number: 2.7.2.11

L-glutamate + ATP <=> γ-L-glutamyl 5-phosphate + ADP

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.

The reaction is irreversible in the direction shown.

Alternative Substrates for L-glutamate: cis-cycloglutamate [Seddon89 ]

In Pathways: proline biosynthesis I

Cofactors or Prosthetic Groups: Mg2+ [PerezArellano05, PerezArellano06, Smith84]

Inhibitors (Allosteric): L-proline [PerezArellano06, Comment 5]

Inhibitors (Competitive): ADP [Smith84]

Primary Physiological Regulators of Enzyme Activity: L-proline , ADP

Kinetic Parameters:

Substrate
Km (μM)
Citations
ATP
2000.0
[PerezArellano06, BRENDA14]
ATP
500.0, 400.0
[Smith84, BRENDA14]

pH(opt): 6.5 [Smith84]


Subunit of: γ-glutamyl kinase-GP-reductase multienzyme complex

Subunit composition of γ-glutamyl kinase-GP-reductase multienzyme complex = [(ProB)4][(ProA)4]
         γ-glutamyl kinase = (ProB)4 (extended summary available)
         glutamate-5-semialdehyde dehydrogenase = (ProA)4 (summary available)


Enzymatic reaction of: γ-glutamyl kinase-GP-reductase multienzyme complex

Synonyms: glutamyl kinase-GSA dehydrogenase multienzyme complex

L-glutamate + ATP + NADPH + H+ <=> ADP + L-glutamate-5-semialdehyde + NADP+ + phosphate

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.

The reaction is irreversible in the direction shown.

Summary:
Glutamyl kinase requires GSA dehydrogenase for activity. [Smith84]


Sequence Features

Feature Class Location Attached Group Citations Comment
Amino-Acid-Sites-That-Bind 10  
[UniProt12b]
UniProt: ATP; Non-Experimental Qualifier: probable.
Amino-Acid-Sites-That-Bind 50  
[UniProt12b]
UniProt: Substrate.
Amino-Acid-Sites-That-Bind 137  
[UniProt12b]
UniProt: Substrate.
Sequence-Conflict 143  
[Takemoto96, Deutch84, UniProt10a]
Alternate sequence: E → A; UniProt: (in Ref. 1 and 2);
Amino-Acid-Sites-That-Bind 149  
[UniProt12b]
UniProt: Substrate; via amide nitrogen.
Nucleotide-Phosphate-Binding-Region 169 -> 170 ATP
[UniProt14]
UniProt: ATP; Non-Experimental Qualifier: probable.
Nucleotide-Phosphate-Binding-Region 211 -> 217 ATP
[UniProt14]
UniProt: ATP; Non-Experimental Qualifier: probable.
Conserved-Region 275 -> 353  
[UniProt09]
UniProt: PUA;


Gene Local Context (not to scale): ?

Transcription Unit:

Notes:

History:
10/20/97 Gene b0242 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10768; 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

Baich65: Baich A, Pierson DJ (1965). "Control of proline synthesis in Escherichia coli." Biochim Biophys Acta 104(2);397-404. PMID: 5322806

Baich69: Baich A (1969). "Proline synthesis in Escherichia coli. A proline-inhibitable glutamic acid kinase." Biochim Biophys Acta 1969;192(3);462-7. PMID: 4904678

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

Csonka88: Csonka LN, Gelvin SB, Goodner BW, Orser CS, Siemieniak D, Slightom JL (1988). "Nucleotide sequence of a mutation in the proB gene of Escherichia coli that confers proline overproduction and enhanced tolerance to osmotic stress." Gene 1988;64(2);199-205. PMID: 2841193

Deutch84: Deutch AH, Rushlow KE, Smith CJ (1984). "Analysis of the Escherichia coli proBA locus by DNA and protein sequencing." Nucleic Acids Res 1984;12(15);6337-55. PMID: 6089111

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

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

MarcoMarin07: Marco-Marin C, Gil-Ortiz F, Perez-Arellano I, Cervera J, Fita I, Rubio V (2007). "A novel two-domain architecture within the amino acid kinase enzyme family revealed by the crystal structure of Escherichia coli glutamate 5-kinase." J Mol Biol 367(5);1431-46. PMID: 17321544

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

PerezArellano04: Perez-Arellano I, Gil-Ortiz F, Cervera J, Rubio V (2004). "Glutamate-5-kinase from Escherichia coli: gene cloning, overexpression, purification and crystallization of the recombinant enzyme and preliminary X-ray studies." Acta Crystallogr D Biol Crystallogr 60(Pt 11);2091-4. PMID: 15502337

PerezArellano05: Perez-Arellano I, Rubio V, Cervera J (2005). "Dissection of Escherichia coli glutamate 5-kinase: functional impact of the deletion of the PUA domain." FEBS Lett 579(30);6903-8. PMID: 16337196

PerezArellano06: Perez-Arellano I, Rubio V, Cervera J (2006). "Mapping active site residues in glutamate-5-kinase. The substrate glutamate and the feed-back inhibitor proline bind at overlapping sites." FEBS Lett 580(26);6247-53. PMID: 17069808

Seddon89: Seddon AP, Zhao KY, Meister A (1989). "Activation of glutamate by gamma-glutamate kinase: formation of gamma-cis-cycloglutamyl phosphate, an analog of gamma-glutamyl phosphate." J Biol Chem 1989;264(19);11326-35. PMID: 2567735

Smith84: Smith CJ, Deutch AH, Rushlow KE (1984). "Purification and characteristics of a gamma-glutamyl kinase involved in Escherichia coli proline biosynthesis." J Bacteriol 1984;157(2);545-51. PMID: 6319365

Takemoto96: Takemoto K., Mori H., Murayama N., Kataoka K., Yano M., Itoh T., Yamamoto Y., Inokuchi H., Miki T., Hatada E., Fukuda R., Ichihara S., Mizuno T., Makino K., Nakata A., Yura T., Sampei G., Mizobuchi K. (1996). "Systematic sequencing of the Escherichia coli genome: analysis of the 4.0 - 6.0 min (189,987 - 281,416bp) region." Data submission to EMBL/GenBank/DDBJ databases on 1996-02.

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

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

UniProt12b: UniProt Consortium (2012). "UniProt version 2012-11 released on 2012-11-26 00:00:00." Database.

UniProt14: UniProt Consortium (2014). "UniProt version 2014-01 released on 2014-01-01 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."

UniProtGOA12: UniProt-GOA (2012). "Gene Ontology annotation based on UniPathway vocabulary mapping."


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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 Sat Nov 22, 2014, biocyc14.