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Information on EC 2.7.2.11 - glutamate 5-kinase and Organism(s) Escherichia coli and UniProt Accession P0A7B5
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2.7.2.11 glutamate 5-kinaseIUBMB Comments
In the absence of downstream enzymes, the product rapidly cyclizes to 5-oxo-L-proline and phosphate.
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Word Map
- 2.7.2.11
- proba
- 1-pyrroline-5-carboxylate
- l-proline
-
osmotolerance
- delta1-pyrroline-5-carboxylate
-
osmoprotection
- gpr
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catharanthus
-
uprooted
- biotechnology
The taxonomic range for the selected organisms is: Escherichia coli
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
gamma-glutamyl kinase, glutamate 5-kinase, glutamate kinase, gamma-gk, glutamate-5-kinase, gamma-glutamate kinase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
ATP-L-glutamate 5-phosphotransferase
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ATP:gamma-L-glutamate phosphotransferase
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gamma-glutamate kinase
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gamma-glutamyl kinase
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gamma-glutamylphosphate kinase
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glutamate kinase
GPK
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kinase (phosphorylating), glutamate
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kinase, glutamate (phosphorylating)
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glutamate kinase
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
phospho group transfer
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PATHWAY SOURCE
PATHWAYS
KEGG
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-, -, -, -
SYSTEMATIC NAME
IUBMB Comments
ATP:L-glutamate 5-phosphotransferase
In the absence of downstream enzymes, the product rapidly cyclizes to 5-oxo-L-proline and phosphate.
CAS REGISTRY NUMBER
COMMENTARY
54596-30-4
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
ATP + cis-cycloglutamate
ADP + cis-cycloglutamyl phosphate
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no reaction with trans-cycloglutamate
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?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
AAK domain is responsible for catalysis
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?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
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?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
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the enzyme catalyzes the first step in the pathway from glutamate to proline
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?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
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enzyme is involved in biosynthesis of proline
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?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
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enzyme catalyzes the first step of proline biosynthesis
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?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
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the enzyme catalyzes the first step in the pathway from glutamate to proline
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-
?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
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enzyme is involved in biosynthesis of proline
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?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
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enzyme catalyzes the first step of proline biosynthesis
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?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Mg2+
the AAK and PUA domains of one subunit associate non-canonically in the dimer with the same domains of the other subunit, leaving a negatively charged hole between them that hosts two Mg ions in one crystal, in line with the G5K requirement for free Mg
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
5-oxoproline
AAK domain has a crater on the beta sheet C-edge that hosts the active centre and binds 5-oxoproline
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.18
L-proline
Escherichia coli
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mutant R25S/E30K,I193A, pH not specified in the publication, 37°C
96
L-proline
Escherichia coli
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mutant E143A/K145A, pH not specified in the publication, 37°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
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two glutamyl kinases of MW 125000 Da and of 38000 Da are detected by gel filtration on Sephadex G-150, a single glutamyl kinase of 250000 Da is detected by Bio-gel A1.5M chromatpgraphy
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
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functional unit of the Escherichia coli enzyme is dimeric and contains an intermolecular hydrogen-bond network that interconnects the active-center cavities of the monomers and is important for substrate binding
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
in about 4-5 months, using the hanging drop vapour diffusion method, complexed with glutamate and sulfate, or with L-glutamate 5-phosphate, sulfate and 5-oxoproline, at 2.9 A and 2.5 A resolution, belongs to the space groups P41212 or P21, respectively. Dimer of dimers architecture, each subunit contains a 257 residue AAK domain, typical of acylphosphate-forming enzymes, with characteristic alpha3beta8alpha4 sandwich topology, each subunit contains a 93 residue C-terminal PUA domain, typical of RNA-modifying enzymes, which presents the characteristic beta5beta4 sandwich fold and three alpha helices
hanging-drop vapour-diffusion method at 21°C in the presence of ADP, MgCl2 and L-glutamate using 1.6 M MgSO4, 0.1 M KCl in 0.1 M MES pH 6.5 as crystallization solution. The tetragonal bipyramid-shaped crystals diffract to 2.5 A resolution using synchrotron radiation. The crystals belong to space group P4(1)(3)2(1)2, with unit-cell parameters a = b = 101.1, c = 178.6 A, and contain two monomers in the asymmetric unit, with 58% solvent content
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
K217A
activity is less than 1% of that of wild-type G5K, decreased proline requirement
N149A
activity is less than 1% of that of wild-type G5K, 14fold increased proline requirement
D137A
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mutation hampers proline binding and glutamate binding, IC50 (L-proline) 142fold increased compared to wild-type
E135A
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mutation of Glu135 and Lys145 only produce relatively small changes in proline activity, IC50 (L-proline) comparable to wild-type
E143A
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mutant shows an 38fold augmented IC0.5 (L-proline) while kinetic parameters of glutamate and ATP are scarcely changed, IC50 (L-proline) 38fold increased compared to wild-type
E143A/K145A
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mutant shows an enhanced affinity for L-glutamate and increased IC50 (L-proline) compared to wild-type
I53A
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decreased kinetic parameters, IC50 (L-proline) increased 5fold compared to wild-type
I69E
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mutation produces a very strong (170fold) decrease on proline activity with no other consequence on the kinetic parameters of the enzyme
K145A
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mutant shows an enhanced affinity for L-glutamate and increased IC50 (L-proline) compared to wild-type
N134D
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mutation hampers proline binding and glutamate binding, IC50 (L-proline) 76fold increased compared to wild-type
Q100A
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mutant shows drastically reduced catalytic rate and reduced affinity for glutamate, IC50 (L-proline) 3fold decreased compared to wild-type
Q80A
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mutant shows drastically reduced catalytic rate and reduced affinity for glutamate, IC50 (L-proline) 3fold decreased compared to wild-type
R118A
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mutant shows increased affinity for glutamate and reduced L-proline affinity (63fold increased IC50)
R25S/E30K/I193A
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mutant behaves as a dimer in gel filtration experiments, kinetically indistinguishable from wild-type, IC50 (L-proline) comparable to wild-type
S50A
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mutant exhibits a greatly reduced catalytic rate but has a small effect on apparent affinities for glutamate or ATP, IC50 (L-proline) 3fold increased compared to wild-type
additional information
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2-amino-acid insertion (Val and Asn) in front of Glu143: insertion mutant exhibits a dramatic reduction in catalytic ability (the velocity at infinite concentration of substrates is 5% relative to wild-type), IC50 (L-proline) is enhanced compared to wild-type
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
from strain BRL806, designated as gamma-glutamyl kinase w+ and from reductase-overproducing strain BRL1945, designated as gamma-glutamyl kinase DHPr
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
Escherichia coli DH5alpha strain proB cloning into pET-22b to yield pGKE, and overexpression
proB cloning into pET-22b to yield pGKE and overexpression in Escherichia coli BL21(DE3)
an artificial bifunctional enzyme, gamma-glutamyl kinase/gamma-glutamyl phosphate reductase obtained by fusing the Escherichia coli genes proA and proB improves NaCl tolerance when expressed in Escherichia coli. The proB gene is fused to the 5'-end of the proA gene with a linker encoding five amino acids
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
biotechnology
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an artificial bifunctional enzyme, gamma-glutamyl kinase/gamma-glutamyl phosphate reductase, improves NaCl tolerance when expressed in Escherichia coli
additional information
additional information
G5K and the homologous acetylglutamate kinase closely resemble each other concerning substrate binding and catalysis, but that they have different mechanisms of feed-back control, roles of K10, K217 and T169 in catalysis and ATP binding and of D150 in orienting the catalytic lysines, roles of D148 and D150 in glutamate binding and of D148 and N149 in proline binding
additional information
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G5K and the homologous acetylglutamate kinase closely resemble each other concerning substrate binding and catalysis, but that they have different mechanisms of feed-back control, roles of K10, K217 and T169 in catalysis and ATP binding and of D150 in orienting the catalytic lysines, roles of D148 and D150 in glutamate binding and of D148 and N149 in proline binding
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Hayzer, D.J.; Moses, V.
The enzymes of proline biosynthesis in Escherichia coli. Their molecular weights and the problem of enzyme aggregation
Biochem. J.
173
219-228
1978
Escherichia coli
Meijer, P.J.; Lilius, G.; Holmberg, N.; Bulow, L
An artificial bifunctional enzyme, gamma-glutamyl kinase/gamma-glutamyl phosphate reductase, improves NaCl tolerance when expressed in Escherichia Coli
Biotechnol. Lett.
18
1133-1138
1996
Escherichia coli
-
Smith, C.J.; Deutch, A.H.; Rushlow, K.E.
Purification and characteristics of a gamma-glutamyl kinase involved in Escherichia coli proline biosynthesis
J. Bacteriol.
157
545-551
1984
Escherichia coli
Seddon, A.P.; Zhao, K.Y.; Meister, A.
Activation of glutamate by gamma-glutamate kinase: formation of gamma-cis-cycloglutamyl phosphate, an analog of gamma-glutamyl phosphate
J. Biol. Chem.
264
11326-11335
1989
Escherichia coli, Escherichia coli CM 25
Perez-Arellano, I.; Gil-Ortiz, F.; Cervera, J.; Rubio, V.
Glutamate-5-kinase from Escherichia coli: gene cloning, overexpression, purification and crystallization of the recombinant enzyme and preliminary X-ray studies
Acta Crystallogr. Sect. D
60
2091-2094
2004
Escherichia coli
Perez-Arellano, I.; Rubio, V.; Cervera, J.
Mapping active site residues in glutamate-5-kinase. The substrate glutamate and the feed-back inhibitor proline bind at overlapping sites
FEBS Lett.
580
6247-6253
2006
Escherichia coli (P0A7B5), Escherichia coli
Marco-Marin, C.; Gil-Ortiz, F.; Perez-Arellano, I.; Cervera, J.; Fita, I.; Rubio, V.
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
1431-1446
2007
Escherichia coli (P0A7B5), Escherichia coli
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