Information on EC 2.7.2.11 - glutamate 5-kinase

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY
2.7.2.11
-
RECOMMENDED NAME
GeneOntology No.
glutamate 5-kinase
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
ATP + L-glutamate = ADP + L-glutamate 5-phosphate
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
phospho group transfer
-
-
-
-
PATHWAY
KEGG Link
MetaCyc Link
Arginine and proline metabolism
-
citrulline biosynthesis
-
Metabolic pathways
-
ornithine de novo biosynthesis
-
proline biosynthesis I
-
SYSTEMATIC NAME
IUBMB Comments
ATP:L-glutamate 5-phosphotransferase
Product rapidly cyclizes to 5-oxoproline and phosphate.
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
ATP-L-glutamate 5-phosphotransferase
-
-
-
-
ATP:gamma-L-glutamate phosphotransferase
-
-
-
-
G5K
P0A7B5
-
gamma-GK
Q6E235
-
gamma-GK
Bacillus subtilis 93151-14
Q6E235
-
-
gamma-glutamate kinase
-
-
-
-
gamma-glutamyl kinase
-
-
-
-
gamma-glutamyl kinase
Q6E235
-
gamma-glutamyl kinase
Bacillus subtilis 93151-14
Q6E235
-
-
gamma-glutamyl kinase
-
-
gamma-glutamyl kinase
-
-
gamma-glutamyl kinase
P32264
-
gamma-glutamyl kinase
Saccharomyces cerevisiae INVDput1pro1
P32264
-
-
gamma-glutamyl kinase
-
-
gamma-glutamylphosphate kinase
-
-
-
-
GK
Saccharomyces cerevisiae INVDput1pro1
P32264
-
-
glutamate kinase
-
-
-
-
glutamate kinase
-
-
glutamate kinase
-
-
glutamate kinase
-
-
glutamate-5-kinase
-
-
GPK
-
-
-
-
kinase (phosphorylating), glutamate
-
-
-
-
kinase, glutamate (phosphorylating)
-
-
-
-
proB
-
gene name
CAS REGISTRY NUMBER
COMMENTARY
54596-30-4
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
strain 93151-14
SwissProt
Manually annotated by BRENDA team
Bacillus subtilis 93151-14
strain 93151-14
SwissProt
Manually annotated by BRENDA team
strain CM 25
-
-
Manually annotated by BRENDA team
Escherichia coli CM 25
strain CM 25
-
-
Manually annotated by BRENDA team
strain PAO 1
-
-
Manually annotated by BRENDA team
Pseudomonas aeruginosa PAO 1
strain PAO 1
-
-
Manually annotated by BRENDA team
strain INVDput1pro1
SwissProt
Manually annotated by BRENDA team
Saccharomyces cerevisiae INVDput1pro1
strain INVDput1pro1
SwissProt
Manually annotated by BRENDA team
var. Ailsa Craig
-
-
Manually annotated by BRENDA team
cv. Matador
-
-
Manually annotated by BRENDA team
cv. Mironovska 808
-
-
Manually annotated by BRENDA team
salt tolerant cultivar T-44 and salt sensitive variant SML-32
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
physiological function
-
Chlamydomonas reinhardtii gamma-glutamte kinase is functional as demonstrated by successful complementation of Escherichia coli auxotroph JW0232 (strain in which the proB gene has been knocked-out)
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + 5-ethyl-L-glutamate
?
show the reaction diagram
Pseudomonas aeruginosa, Pseudomonas aeruginosa PAO 1
-
5% of the activity with L-glutamate
-
-
?
ATP + 5-methyl-L-glutamate
?
show the reaction diagram
Pseudomonas aeruginosa, Pseudomonas aeruginosa PAO 1
-
6% of the activity with L-glutamate
-
-
?
ATP + cis-cycloglutamate
ADP + cis-cycloglutamyl phosphate
show the reaction diagram
Escherichia coli, Escherichia coli CM 25
-
no reaction with trans-cycloglutamate
-
?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
show the reaction diagram
-
-
-
-
?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
show the reaction diagram
-
-
-
-
?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
show the reaction diagram
P0A7B5
-
-
-
?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
show the reaction diagram
-
-
-
-
?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
show the reaction diagram
P32264
-
-
-
?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
show the reaction diagram
-
-
-
-
?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
show the reaction diagram
-
-
-
-
?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
show the reaction diagram
-
-
-
-
?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
show the reaction diagram
-
-
-
-
?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
show the reaction diagram
-
-
-
-
?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
show the reaction diagram
-
-
-
-
?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
show the reaction diagram
-
-
-
-
?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
show the reaction diagram
P32264
-
-
-
?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
show the reaction diagram
-, Q6E235
-
-
-
?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
show the reaction diagram
-
the enzyme catalyzes the first step in the pathway from glutamate to proline
-
-
?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
show the reaction diagram
-
enzyme GK 1 is the first enzyme of the proline biosynthetic pathway
-
-
?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
show the reaction diagram
-
enzyme is involved in biosynthesis of proline
-
-
?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
show the reaction diagram
-
enzyme form GK1 is involved in biosynthesis of L-Pro, enzyme form GK 2 is involved in biosynthesis of glutamine and the function of enzyme form GK 3 has not been found
-
-
?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
show the reaction diagram
-
enzyme catalyzes the first step of proline biosynthesis
-
-
?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
show the reaction diagram
-
AAK domain is responsible for catalysis
-
-
?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
show the reaction diagram
Saccharomyces cerevisiae INVDput1pro1
P32264
-
-
-
?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
show the reaction diagram
Bacillus subtilis 93151-14
Q6E235
-
-
-
?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
show the reaction diagram
Escherichia coli CM 25
-
-, the enzyme catalyzes the first step in the pathway from glutamate to proline
-
-
?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
show the reaction diagram
Pseudomonas aeruginosa PAO 1
-
-
-
-
?
ATP + L-glutamine
?
show the reaction diagram
Pseudomonas aeruginosa, Pseudomonas aeruginosa PAO 1
-
10% of the activity with L-glutamate
-
-
?
GTP + L-glutamate
GDP + L-glutamate 5-phosphate
show the reaction diagram
Pseudomonas aeruginosa, Pseudomonas aeruginosa PAO 1
-
10% of the activity with ATP
-
-
?
additional information
?
-
-
relation of glutamate kinase activity to cadmium and zinc concentration in the above-ground biomass, regulatory role in plant heavy metal stress adaptation
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
show the reaction diagram
-
-
-
-
?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
show the reaction diagram
-
the enzyme catalyzes the first step in the pathway from glutamate to proline
-
-
?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
show the reaction diagram
-
enzyme GK 1 is the first enzyme of the proline biosynthetic pathway
-
-
?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
show the reaction diagram
-
enzyme is involved in biosynthesis of proline
-
-
?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
show the reaction diagram
-
enzyme form GK1 is involved in biosynthesis of L-Pro, enzyme form GK 2 is involved in biosynthesis of glutamine and the function of enzyme form GK 3 has not been found
-
-
?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
show the reaction diagram
-
enzyme catalyzes the first step of proline biosynthesis
-
-
?
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
show the reaction diagram
Escherichia coli CM 25
-
the enzyme catalyzes the first step in the pathway from glutamate to proline
-
-
?
METALS and IONS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
Cd2+
-
increased content in soil decreases enzyme activity
K+
-
the enzyme is most active at 30C at a relative high K+ + Na+ concentration and a K+/Na+ ratio of 1.8 to 10.2 and at 0C at both a lower K+ + Na+ concentration and a K+/Na+ ratio
Mg2+
-
required
Mg2+
-
required
Mg2+
-
enzyme GK 1 is strongly activated by Mg2+, maximum at 60 mM Mg2+
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
Mn2+
-
can partially replace Mg2+
Na+
-
the enzyme is most active at 30C at a relative high K+ + Na+ concentration and a K+/Na+ ratio of 1.8 to 10.2 and at 0C at both a lower K+ + Na+ concentration and a K+/Na+ ratio
Zn2+
-
increased content in soil decreases enzyme activity
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
5-oxo-L-Pro
-
12 mM, 10% inhibition
5-oxoproline
-
AAK domain has a crater on the beta sheet C-edge that hosts the active centre and binds 5-oxoproline
As2+
-
applied into soil strongly decreases activity by 87.7%
Cd2+
-
0.1 mM, complete inhibition
Cd2+
-
applied into soil strongly decreases activity
Cd2+
-
increased content in soil decreases enzyme activity
cis-4-hydroxy-L-proline
-
-
DL-3,4-Didehydroproline
-
9 mM, 50% inhibition
Hg2+
-
0.1 mM, complete inhibition
iodoacetamide
-
0.125 mM, 60% inhibition. Preincubation with 0.25 mM dithiothreitol for 5 min partially protects
L-azetidine-2-carboxylic acid
-
3 mM, 50% inhibition
L-methionine-DL-sulphoximine
-
competitive with L-glutamate
L-Orn
-
12 mM, 10% inhibition
L-Pro
-
enzyme from strain PAO1: 5 mM, 50% inhibition, complete inhibition at 30 mM, noncompetitive. Strain PAO 879, a proline-auxotroph mutant lacks a proline-inhibitable gamma-glutamyl kinase
L-Pro
-
IC50: 0.08 mM, at room temperature. At low temperatures the inhibition switches over into allosteric activation and the biosynthesis of proline is started
L-Pro
-
feedback-inhibition
L-proline
-, Q6E235
completely inhibits the activity of wild-type gamma-glutamyl kinase/gamma-glutamyl phosphate reductase at concentrations greater than 0.1mM, and inhibits it by 30% at 0.01mM
L-proline
P32264
GK activity is subject to feedback inhibition by proline
L-proline
-
AAK domain is responsible for inhibition
L-proline
-
free proline decreases activity, allosteric regulation
L-proline
-
results indicate that the sensitivity of Chlamydomonas glutamte kinase to feedback inhibition by L-proline is much lower than that of similar microbial glutamte kinase
L-Thioproline
-
12 mM, 10% inhibition
methyl L-proline
-
-
-
NEM
-
0.125 mM, complete inhibition. Preincubation with 0.25 mM dithiothreitol for 5 min partially protects
PCMB
-
0.125 mM, complete inhibition. Preincubation with 0.25 mM dithiothreitol for 5 min partially protects
trans-4-hydroxy-L-proline
-
-
Zn2+
-
applied into soil strongly decreases activity
Zn2+
-
increased content in soil decreases enzyme activity
additional information
P32264
no significant differences in growth between mutant yeast cells on SD agar medium with heat shock (50C, 3 to 5 h), ethanol (16 to20%), and sorbitol (2.0 to 2.5 M) stresses, although the proline-accumulating cells are more resistant to these stresses than cells expressing wild-type GK
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
L-Pro
-
at low temperatures the inhibition switches over into allosteric activation and the biosynthesis of proline is started
additional information
P32264
GK activity requires gamma-glutamyl phosphate reductase
-
additional information
-
significant increase of the glutamate kinase activity in the spinach biomass growing on sewage sludge treatment
-
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.4
-
ATP
-
pH 7.0, 37C, gamma-glutamyl kinase DHPr
0.5
-
ATP
-
pH 7.0, 37C, gamma-glutamyl kinase w+
1.8
-
ATP
-
mutant D170N
2
-
ATP
-
wild-type
2.3
-
ATP
-
mutant D148N
3.4
-
ATP
-
mutant D170A
4.2
-
ATP
-
mutant D150N
4.3
-
ATP
-
mutant G51A
4.5
-
ATP
-
mutant N149A
6.1
-
ATP
-
mutant K10A; mutant T169S
6.4
-
ATP
-
mutant M214A
6.5
-
ATP
-
mutant D148A
17.6
-
ATP
-
mutant K217R
18.4
-
ATP
-
mutant T169A
24.6
-
ATP
-
mutant K217A
12
-
L-glutamate
-
-
Ki VALUE [mM]
Ki VALUE [mM] Maximum
INHIBITOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.06
-
ADP
-
pH 7.0, 37C
0.09
-
L-Pro
-
wild-type enzyme
1.9
-
L-Pro
-
mutant enzyme I79T
17
-
L-Pro
-
mutant enzyme A62V
19
-
L-Pro
-
mutant enzyme S159P
20
-
L-Pro
-
mutant enzyme I149F
23
-
L-Pro
-
mutant enzyme M94T
50
-
L-Pro
-
mutant enzyme E153A or E153G
55
-
L-Pro
-
mutant enzyme D162G
58
-
L-Pro
-
mutant enzyme D162N
82
-
L-Pro
-
mutant enzyme A62T
90
-
L-Pro
-
mutant enzyme L154S
180
-
L-Pro
-
mutant enzyme D147G
310
-
L-Pro
-
mutant enzyme E153K
IC50 VALUE [mM]
IC50 VALUE [mM] Maximum
INHIBITOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.08
-
L-Pro
-
IC50: 0.08 mM, at room temperature. At low temperatures the inhibition switches over into allosteric activation and the biosynthesis of proline is started
0.011
-
L-proline
-
mutant T169A
0.016
-
L-proline
-
mutant K217A
0.02
-
L-proline
-
mutant K217R
0.021
-
L-proline
-
mutant T169S
0.05
-
L-proline
-
mutant Q100A, pH not specified in the publication, 37C; mutant Q80A, pH not specified in the publication, 37C
0.14
-
L-proline
-
mutant E135A, pH not specified in the publication, 37C
0.15
-
L-proline
-
wild-type
0.15
-
L-proline
-
wild-type, pH not specified in the publication, 37C
0.165
-
L-proline
-
mutant M214A
0.17
-
L-proline
-
mutant D170A
0.176
-
L-proline
-
mutant D150N
0.18
-
L-proline
-
mutant R25S/E30K,I193A, pH not specified in the publication, 37C
0.22
-
L-proline
-
mutant D170N
0.26
-
L-proline
-
mutant K10A
0.5
-
L-proline
P32264
wild-type
0.5
-
L-proline
-
mutant S50A, pH not specified in the publication, 37C
0.51
-
L-proline
-
mutant G51A
1.2
-
L-proline
-
mutant I53A, pH not specified in the publication, 37C
1.3
-
L-proline
-
mutant K145A, pH not specified in the publication, 37C
2.16
-
L-proline
-
mutant N149A
5.7
-
L-proline
-
mutant E143A, pH not specified in the publication, 37C
5.9
-
L-proline
-
mutant D148N
6.1
-
L-proline
-
mutant D107A, pH not specified in the publication, 37C
9.4
-
L-proline
-
mutant R118A, pH not specified in the publication, 37C
11.5
-
L-proline
-
mutant N134D, pH not specified in the publication, 37C
21
-
L-proline
-
mutant D148A
21.4
-
L-proline
-
mutant D137A, pH not specified in the publication, 37C
26
-
L-proline
-
mutant I69E, pH not specified in the publication, 37C
32
-
L-proline
P32264
mutant D154N
50
-
L-proline
P32264
mutant I150T
54
-
L-proline
P32264
mutant N142D/I166V
90
-
L-proline
-
pH 7.0, 37C
96
-
L-proline
-
mutant E143A/K145A, pH not specified in the publication, 37C
840
-
L-proline
P32264
mutant E149K
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
3.2
-
P32264
mutant N142D/I166V
4.2
-
P32264
mutant E149K
14.7
-
P32264
wild-type
26.3
-
P32264
mutant D154N
29
-
P32264
mutant I150T
additional information
-
-
-
additional information
-
-
relative changes of glutamate kinase activity in above-ground biomass determined, changes of enzyme activity suggests regulatory role in plant heavy metal stress adaptation, potential use as a stress biomarker, toxic effects of cadmium and zinc tested at different levels, significant decrease of enzyme activity in plants grown on contaminated treatments, decline in period of damage of cell activities, increase of activity in period of maximum resistance and following decrease in period of the plant metabolism depletion
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
7.5
-
-
assay at
7.6
-
-
activity assay at, 50 mM phosphate buffer
pH RANGE
pH RANGE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
5.5
8.5
-
pH 5.5: about 80% of maximal activity, pH 8.5: about 40% of maximal activity
6
7.5
-
50% of maximal activity at pH 6.0 and at pH 7.5
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
37
-
-
assay at
SOURCE TISSUE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
SOURCE
-
three glutamate kinases: GK 1, GK 2, and GK 3
Manually annotated by BRENDA team
-
above-ground biomass
Manually annotated by BRENDA team
-
2.3-fold increase in presence of 200 mM NaCl in cultivar T-44, while in the presence of 50 mM NaCl in cultivar SML-32 increase is 1.1-fold on day 5 as compared to non-saline control. Shoot maintain higher activity than the root
Manually annotated by BRENDA team
-
above-ground biomass
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
-
computer predictions suggest that a chloroplast transit peptide exists at the N-terminal region
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
Burkholderia thailandensis (strain E264 / ATCC 700388 / DSM 13276 / CIP 106301)
Campylobacter jejuni subsp. jejuni serotype O:2 (strain NCTC 11168)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
42000
-
-
SDS-PAGE
47000
-
P32264
gel filtration
84000
-
-
gel filtration
100000
-
-
SDS-PAGE, recombinant protein
236000
-
-
gamma-glutamyl kinase DHPr, gel filtration
254000
-
-
gel filtration
additional information
-
-
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
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
?
-
x * 41984, calculation from nucleotide sequence
?
-
x * 42000 + x * 84000, gel filtration after dissociation into subunits
dimer
-
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
hexamer
-
6 * 40000, gamma-glutamyl kinase DHPr, SDS-PAGE
tetramer
-
crystallographic studies
tetramer
-
gel filtration, 4 * 42000
tetramer
-
glutamte kinase crystalizes as a tetramer
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
hanging-drop vapour-diffusion method at 21C 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
-
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
-
pH STABILITY
pH STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
TEMPERATURE STABILITY
TEMPERATURE STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
70
85
-
the enzyme retains approximately 100, 90 and 65% of its activity when heated for 45 min at 70, 80 and 85C, respectively
90
-
-
at 90C, tmGK displays around 40% of the activity when heated for 45 min
STORAGE STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
-70C, stable for several months
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-40C, 40% loss of activity within 2 months, then remains stable
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Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
using Ni-NTA chromatography
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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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to homogeneity
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by Ni2+ column and gel filtration
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using Ni-NTA chromatography
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(NH4)2SO4 precipitation and a single ion-exchange chromatography step are used
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enzyme form GK 1
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Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
proBA fusion gene and mutated proBA fusion gene expressed in Escherichia coli JM83
-, Q6E235
expressed in Escherichia coli as a His-tagged fusion protein
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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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cloned in pET22 and overexpressed in Escherichia coli
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Escherichia coli DH5alpha strain proB cloning into pET-22b to yield pGKE, and overexpression
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proB cloning into pET-22b to yield pGKE and overexpression in Escherichia coli BL21(DE3)
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expressed in Escherichia coli as a His-tagged fusion protein
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expression of PRO1 in Escherichia coli M15(pREP4)
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expression in Escherichia coli
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expressed in Escherichia coli as a His-tagged fusion protein
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ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
N177D
-, Q6E235
mutant of the proBA fusion gene, improves the osmotolerance of host cells of Escherichia coli JM83, leads to overproduction of proline by host cells, is about 100fold less sensitive to proline-mediated feedback inhibition than the control
N177D
Bacillus subtilis 93151-14
-
mutant of the proBA fusion gene, improves the osmotolerance of host cells of Escherichia coli JM83, leads to overproduction of proline by host cells, is about 100fold less sensitive to proline-mediated feedback inhibition than the control
-
D107A
-
mutant shows 40fold increased IC50 (L-proline)
D137A
-
mutation hampers proline binding and glutamate binding, IC50 (L-proline) 142fold increased compared to wild-type
D148A
-
is active, 150fold increased proline requirement
D148N
-
is active, 40fold increased proline requirement
D150A
-
is not active
D150N
-
is active
D170A
-
activity is less than 1% of that of wild-type G5K
D170N
-
is active
E135A
-
mutation of Glu135 and Lys145 only produce relatively small changes in proline activity, IC50 (L-proline) comparable to wild-type
E143A
-
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
-
mutant shows an enhanced affinity for L-glutamate and increased IC50 (L-proline) compared to wild-type
G51A
-
is active
I53A
-
decreased kinetic parameters, IC50 (L-proline) increased 5fold compared to wild-type
I69E
-
mutation produces a very strong (170fold) decrease on proline activity with no other consequence on the kinetic parameters of the enzyme
K10A
-
activity is less than 1% of that of wild-type G5K
K145A
-
mutant shows an enhanced affinity for L-glutamate and increased IC50 (L-proline) compared to wild-type
K217A
-
activity is less than 1% of that of wild-type G5K, decreased proline requirement
K217R
-
is active, decreased proline requirement
N134D
-
mutation hampers proline binding and glutamate binding, IC50 (L-proline) 76fold increased compared to wild-type
N149A
-
activity is less than 1% of that of wild-type G5K, 14fold increased proline requirement
Q100A
-
mutant shows drastically reduced catalytic rate and reduced affinity for glutamate, IC50 (L-proline) 3fold decreased compared to wild-type
Q80A
-
mutant shows drastically reduced catalytic rate and reduced affinity for glutamate, IC50 (L-proline) 3fold decreased compared to wild-type
R118A
-
mutant shows increased affinity for glutamate and reduced L-proline affinity (63fold increased IC50)
R25S/E30K/I193A
-
mutant behaves as a dimer in gel filtration experiments, kinetically indistinguishable from wild-type, IC50 (L-proline) comparable to wild-type
S50A
-
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
T169A
-
is active, decreased proline requirement
T169S
-
is active, decreased proline requirement
D154N
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the mutation results in a prominant increase in cell viability after freezing at -20C compared to the viability of the cells harboring the wild-type PRO1 gene. The altered gamma-glutamyl kinase results in stabilization of the complex with gamma-glutamyl phosphate reductase or has an indirect effect on gamma-glutamyl phosphate reductase activity which leads to an increase in L-proline production in Saccharomyces cerevisiae
D154N
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is 64fold less sensitive to feedback inhibition than wild-type GK, leading to proline accumulation
E149K
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very insensitive to feedback inhibition, shows prominent increase in the proline content, freeze tolerance like the D154N mutant
N142D/I166V
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very insensitive to feedback inhibition, shows prominent increase in the proline content, is more tolerant to freezing stress than cells expressing the D154N mutant
S146P
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shows prominent increase in the proline content
D154N
Saccharomyces cerevisiae INVDput1pro1
-
is 64fold less sensitive to feedback inhibition than wild-type GK, leading to proline accumulation
-
E149K
Saccharomyces cerevisiae INVDput1pro1
-
very insensitive to feedback inhibition, shows prominent increase in the proline content, freeze tolerance like the D154N mutant
-
I150T
Saccharomyces cerevisiae INVDput1pro1
-
very insensitive to feedback inhibition, shows prominent increase in the proline content, is more tolerant to freezing stress than cells expressing the D154N mutant
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N142D/I166V
Saccharomyces cerevisiae INVDput1pro1
-
very insensitive to feedback inhibition, shows prominent increase in the proline content, is more tolerant to freezing stress than cells expressing the D154N mutant
-
S146P
Saccharomyces cerevisiae INVDput1pro1
-
shows prominent increase in the proline content
-
A62T
-
drastic reduction in specific activity. 911fold increase in Ki-value for L-Pro compared to wild-type enzyme
A62V
-
drastic reduction in specific activity. 188fold increase in Ki-value for L-Pro compared to wild-type enzyme
D147G
-
reduction in catalytic activity. 2000fold increase in Ki-value for L-Pro compared to wild-type enzyme
D162G
-
drastic reduction in specific activity. 611.1fold increase in Ki-value for L-Pro compared to wild-type enzyme
D162N
-
drastic reduction in specific activity. 644fold increase in Ki-value for L-Pro compared to wild-type enzyme
E153A
-
reduction in catalytic activity. 555.5fold increase in Ki-value for L-Pro compared to wild-type enzyme
E153G
-
reduction in catalytic activity. 555.5fold increase in Ki-value for L-Pro compared to wild-type enzyme
E153K
-
reduction in catalytic activity. 3444.4fold increase in Ki-value for L-Pro compared to wild-type enzyme
I149F
-
222.2fold increase in Ki-value for L-Pro compared to wild-type enzyme; drastic reduction in specific activity. 222.2fold increase in Ki-value for L-Pro compared to wild-type enzyme
I79T
-
reduction in catalytic activity. 21.1fold increase in Ki-value for L-Pro compared to wild-type enzyme
L154S
-
reduction in catalytic activity. 1000fold increase in Ki-value for L-Pro compared to wild-type enzyme
M94T
-
reduction in catalytic activity. 255.6fold increase in Ki-value for L-Pro compared to wild-type enzyme
S159P
-
reduction in catalytic activity. 211.1fold increase in Ki-value for L-Pro compared to wild-type enzyme
D192G
-
the mutation causes an enhanced feedback-resistant gamma-glutamyl kinase activity and conferrs an analogue-resistant phenotype to an Escherichia coli transformant containing the mutated gene
M214A
-
is active
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
I150T
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very insensitive to feedback inhibition, shows prominent increase in the proline content, is more tolerant to freezing stress than cells expressing the D154N mutant
additional information
-
truncated yeast gamma-glutamyl kinase proteins are engineered from which the C-terminal region is deleted. A complementation test in Escherichia coli and yeast and enzymatic analysis of recombinant proteins reveal that a 67-residue linker sequence between a 255-residue kinase domain and a 106-residue archaeosine transglycosylase (PUA) domain is essential for gamma-glutamyl kinase activity. 67 or more residues of the C-terminal region, not the PUA domain itself, are required for the full enzymatic activity
APPLICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
additional information
-, Q6E235
proBA fusion gene and some of the mutated proBA fusion genes can be expressed and functionally complement the proline auxotrophy in Escherichia coli JM83, the fused gamma-glutamyl kinase/gamma-glutamyl phosphate reductase is more active than the separate gamma-glutamyl kinase and gamma-glutamyl phosphate reductase enzymes
additional information
Bacillus subtilis 93151-14
-
proBA fusion gene and some of the mutated proBA fusion genes can be expressed and functionally complement the proline auxotrophy in Escherichia coli JM83, the fused gamma-glutamyl kinase/gamma-glutamyl phosphate reductase is more active than the separate gamma-glutamyl kinase and gamma-glutamyl phosphate reductase enzymes
-
biotechnology
-
an artificial bifunctional enzyme, gamma-glutamyl kinase/gamma-glutamyl phosphate reductase, improves NaCl tolerance when expressed in Escherichia coli
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
biotechnology
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the D154N mutation results in a prominant increase in cell viability after freezing at -20C compared to the viability of the cells harboring the wild-type PRO1 gene, method for breeding novel freeze-tolerant yeast strains
additional information
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GK is the rate-limiting enzyme in the proline biosynthesis
additional information
Saccharomyces cerevisiae INVDput1pro1
-
GK is the rate-limiting enzyme in the proline biosynthesis
-