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Escherichia coli K-12 substr. MG1655 Enzyme: 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)

Gene-Reaction Schematic: ?


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]


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

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

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

Locations: cytosol

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

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

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

GO Terms:

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

MultiFun Terms: metabolism biosynthesis of building blocks amino acids proline

Unification Links: EcoliWiki:b0242 , ModBase:P0A7B5 , PR:PRO_000023597 , Pride:P0A7B5 , Protein Model Portal:P0A7B5 , RefSeq:NP_414777 , 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

Catalyzes:
L-glutamate + ATP → γ-L-glutamyl 5-phosphate + ADP

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]

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]

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

Synonyms: pro(1), pro1, γ-glutamyl phosphate reductase, GPR

Gene: proA Accession Numbers: EG10767 (EcoCyc), b0243, ECK0244

Locations: cytosol

Subunit composition of glutamate-5-semialdehyde dehydrogenase = [ProA]4

Map Position: [260,727 -> 261,980] (5.62 centisomes)
Length: 1254 bp / 417 aa

Molecular Weight of Polypeptide: 44.63 kD (from nucleotide sequence)

GO Terms:

Biological Process: GO:0006561 - proline biosynthetic process Inferred from experiment Inferred by computational analysis [UniProtGOA11, GOA06, GOA01, Deutch84]
GO:0008152 - metabolic process Inferred by computational analysis [GOA01]
GO:0008652 - cellular amino acid biosynthetic process Inferred by computational analysis [UniProtGOA11]
GO:0055114 - oxidation-reduction process Inferred by computational analysis [UniProtGOA11, GOA01]
GO:0055129 - L-proline biosynthetic process Inferred by computational analysis [UniProtGOA12]
Molecular Function: GO:0004350 - glutamate-5-semialdehyde dehydrogenase activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01a, GOA01, Hayzer82, Hayzer81, Hayzer83]
GO:0050661 - NADP binding Inferred from experiment Inferred by computational analysis [GOA01, Hayzer81]
GO:0016491 - oxidoreductase activity Inferred by computational analysis [UniProtGOA11, GOA01]
GO:0016620 - oxidoreductase activity, acting on the aldehyde or oxo group of donors, NAD or NADP as acceptor Inferred by computational analysis [GOA01]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08, LopezCampistrou05]
GO:0005737 - cytoplasm Inferred by computational analysis [UniProtGOA11a, UniProtGOA11, GOA06]

MultiFun Terms: metabolism biosynthesis of building blocks amino acids proline

Unification Links: DIP:DIP-10568N , EcoliWiki:b0243 , Mint:MINT-1261639 , ModBase:P07004 , PR:PRO_000023596 , Pride:P07004 , Protein Model Portal:P07004 , RefSeq:NP_414778 , SMR:P07004 , String:511145.b0243 , Swiss-Model:P07004 , UniProt:P07004

Relationship Links: InterPro:IN-FAMILY:IPR000965 , InterPro:IN-FAMILY:IPR012134 , InterPro:IN-FAMILY:IPR015590 , InterPro:IN-FAMILY:IPR016161 , InterPro:IN-FAMILY:IPR016162 , InterPro:IN-FAMILY:IPR016163 , InterPro:IN-FAMILY:IPR020593 , Panther:IN-FAMILY:PTHR11063:SF1 , Pfam:IN-FAMILY:PF00171 , Prosite:IN-FAMILY:PS01223

Catalyzes:
L-glutamate-5-semialdehyde + NADP+ + phosphate ← γ-L-glutamyl 5-phosphate + NADPH + H+

Summary:
Glutamate-semialdehyde dehydrogenase catalyzes the second reaction, the reduction of γ-glutamyl phosphate to glutamate 5-semialdehyde, in proline biosynthesis I [Hayzer81], [Hayzer82], [Hayzer83]. Due to highly labile nature of γ-L-glutamyl 5-phosphate, the enzyme activity is measured in vitro in the reverse direction. Inhibition and kinetic studies deal with the overall reaction including the spontaneous production of pyrroline-5'-carboxylate.

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

Citations: [Baich71]

Gene Citations: [Deutch84]

Essentiality data for proA 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]

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

Baich71: Baich A (1971). "The biosynthesis of proline in Escherichia coli: phosphate-dependent glutamate -semialdehyde dehydrogenase (NADP), the second enzyme in the pathway." Biochim Biophys Acta 244(1);129-34. PMID: 4399189

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, 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.

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

Hayzer81: Hayzer DJ, Leisinger T (1981). "Proline biosynthesis in Escherichia coli. Stoichiometry and end-product identification of the reaction catalysed by glutamate semialdehyde dehydrogenase." Biochem J 197(2);269-74. PMID: 7034716

Hayzer82: Hayzer DJ, Leisinger T (1982). "Proline biosynthesis in Escherichia coli. Purification and characterisation of glutamate-semialdehyde dehydrogenase." Eur J Biochem 1982;121(3);561-5. PMID: 7035170

Hayzer83: Hayzer DJ, Leisinger T (1983). "Proline biosynthesis in Escherichia coli. Kinetic and mechanistic properties of glutamate semialdehyde dehydrogenase." Biochim Biophys Acta 742(2);391-8. PMID: 6337636

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

LopezCampistrou05: Lopez-Campistrous A, Semchuk P, Burke L, Palmer-Stone T, Brokx SJ, Broderick G, Bottorff D, Bolch S, Weiner JH, Ellison MJ (2005). "Localization, annotation, and comparison of the Escherichia coli K-12 proteome under two states of growth." Mol Cell Proteomics 4(8);1205-9. PMID: 15911532

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

UniProtGOA11: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

UniProtGOA11a: UniProt-GOA (2011). "Gene Ontology annotation based on the manual assignment of UniProtKB Subcellular Location terms 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 Thu Nov 27, 2014, BIOCYC14B.