Information on EC 2.6.1.85 - aminodeoxychorismate synthase

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

EC NUMBER
COMMENTARY hide
2.6.1.85
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RECOMMENDED NAME
GeneOntology No.
aminodeoxychorismate synthase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
chorismate + L-glutamine = 4-amino-4-deoxychorismate + L-glutamate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
amino group transfer
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-
-
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nucleophilic substitution
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
4-aminobenzoate biosynthesis
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Folate biosynthesis
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tetrahydrofolate metabolism
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SYSTEMATIC NAME
IUBMB Comments
chorismate:L-glutamine aminotransferase
The enzyme is composed of two parts, PabA and PabB. In the absence of PabA and glutamine, PabB converts ammonia and chorismate into 4-amino-4-deoxychorismate (in the presence of Mg2+). PabA converts glutamine into glutamate only in the presence of stoichiometric amounts of PabB. This enzyme is coupled with EC 4.1.3.38, aminodeoxychorismate lyase, to form 4-aminobenzoate.
CAS REGISTRY NUMBER
COMMENTARY hide
132264-37-0
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
PabB heterologously overexpressed in Escherichia coli
SwissProt
Manually annotated by BRENDA team
ISP5230 strain
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Manually annotated by BRENDA team
ISP5230 strain
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Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2-amino-2-deoxychorismate + NH3
4-amino-4-deoxychorismate + NH3
show the reaction diagram
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PabB, second half of the reaction, starting with the intermediate
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-
?
chorismate + L-asparagine
4-amino-4-deoxychorismate + L-aspartate
show the reaction diagram
11% of the activity with L-glutamine
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-
?
chorismate + L-glutamine
4-amino-4-deoxychorismate + L-glutamate
show the reaction diagram
chorismate + L-glutamine
L-glutamate + 4-amino-4-deoxychorismate
show the reaction diagram
chorismate + NH3
4-amino-4-deoxychorismate + ?
show the reaction diagram
chorismate + NH3
4-amino-4-deoxychorismate + H2O
show the reaction diagram
chorismate + NH4+
4-amino-4-deoxychorismate + H2O + H+
show the reaction diagram
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-
-
-
?
additional information
?
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isoform PhzE catalyzes the addition of ammonia to C2 of chorismate, as does anthranilate synthase, yet unlike anthranilate synthase it does not catalyze elimination of pyruvate from enzyme-bound aminodeoxyisochorismate. Reaction follows a random kinetic mechanism, enzyme PhzE can use NH4+ as an alternative nucleophile. PhzE shows no lyase activity
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
chorismate + L-glutamine
4-amino-4-deoxychorismate + L-glutamate
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(5R,6R)-4-ammonio-5-((1-carboxylatovinyl)oxy)-6-hydroxycyclohex-1-ene-1-carboxylate
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transition-state analogue, competitive
1-hydroxyphenazine
inhibitory above 0.200 mM
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2-fluorochorismate
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irreversible, covalent modification of K274
atrop-abyssomicin C
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NH3
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inhibitor of the glutamine amidotransferase reaction (Ki = 5 mM, lowered to 0.27 mM if considered that NH3 and not NH4+ is the true inhibitor (pKa value of 9.24))
phosphate
phosphate buffer competitively inhibits PhzE, about 20% inhibition with 100 mM potassium phosphate at pH 7.0
pyocyanin
inhibitory above 0.250 mM
additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
chorismate
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about 50% activation at 0.002 mM
glutamine
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prevents loss of activity
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0016 - 0.24
chorismate
0.045 - 11
L-glutamine
195
NH3
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NH3-dependent p-aminobenzoate production in the absence of glutamine (assay contains 4-amino-4-desoxychorismate lyase), pH 8.0, 37C
145 - 169
NH4+
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.02
2-amino-2-deoxychorismate
Stenotrophomonas maltophilia
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PabB, second half of the reaction, pH 8.0, 25C
0.000098 - 2.2
chorismate
0.013 - 0.79
L-glutamine
0.12
NH3
Arabidopsis thaliana
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NH3-dependent p-aminobenzoate production in the absence of glutamine (assay contains 4-amino-4-desoxychorismate lyase), pH 8.0, 37C
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
13.3 - 143
chorismate
204
3.4 - 17.5
L-glutamine
102
0.00061
NH3
Arabidopsis thaliana
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NH3-dependent p-aminobenzoate production in the absence of glutamine (assay contains 4-amino-4-desoxychorismate lyase), pH 8.0, 37C
27
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0007 - 0.0056
(5R,6R)-4-ammonio-5-((1-carboxylatovinyl)oxy)-6-hydroxycyclohex-1-ene-1-carboxylate
0.13
2-fluorochorismate
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5
NH3
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inhibitor of the glutamine amidotransferase reaction (Ki lowered to 0.27 mM if considered that NH3 and not NH4+ is the true inhibitor (pKa value of 9.24)), pH 8.0, 37C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45
20% of maximum activtiy
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Stenotrophomonas maltophilia (strain K279a)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
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heterodimer of chorismate aminating subunit and glutamine amidotransferase subunit. Mass of glutamine amidotransferase subunit is 50969 Da, as determined by electrospray mass spectrometry
heterodimer
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PabA-PabB
monomer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging drop vapor diffusion at room temperature
pH 7.8, polyethylene glycol monomethylether 550
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20
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at least stable for 30 min
37
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inactivation. DTT, chorismate or PabA prevent inactivation
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20, Tris-HCl buffer, pH 7.4, 50% glycerol
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-80, 50 mM MOPS, 50 mM KCl, 0.1 mM EDTA, 2 mM DTT, 5 mM MgCl, pH 7.6
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
chromatography on Poros HQ50, HS20 cation exchange chromatography and chromatography on hydroxyapatite
immobilized metal ion affinity chromatography (Co2+), fusion tag removed
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immobilized metal ion affinity chromatography (Ni2+), gel filtration
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protamine sulfate precipitation, ammonium sulfate fractionation, chromatography on Sephacryl S-200, chromatography on DEAE-Sephacel and chromatography on dye-agarose
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streptomycin sulfate precipitation, ammonium sulfate fractionation, chromatography on DEAE-cellulose
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
expression in Escherichia coli and Streptomyces lividans
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expression with His-tag
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His-tagged protein (first 43 amino acid residues deleted) expressed in Escherichia coli BL21-CodonPlus (DE3)-RIL
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pabB, optimized for overexpression in Escherichia coli, His-tagged protein expressed in Escherichia coli BL21(DE3)
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
K213A
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subunit PabB, numbering used is based on the EntC protein from Escherichia coli, reduced activity
K213A/Q147K
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subunit PabB, numbering used is based on the EntC protein from Escherichia coli, strongly reduced activity
K274A
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265fold reduction of kcat-value
K274A/L277R/D299E/N303H/I306L/F334Y/C391G
mutant displaying anthranilate synthase activity, the kcat/Kchoris value for anthranilate formation is 25% of the wild-type aminodeoxychorismate synthase value for 4-amino-4-deoxychorismate production
K274R
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640fold reduction of kcat-value
N213V/L214P/K274A/L277R/D299E/N303H/F334Y/P363N/I367L/C391G/G425A
mutant displaying anthranilate synthase activity, is able to produce both 2-amino-2-deoxyisochorismate and anthranilate
N213V/L214P/R259S/K274A/L277R/N303H/F334Y/S366T/C391G/G254A
mutant displaying anthranilate synthase activity, is able to produce both 2-amino-2-deoxyisochorismate and anthranilate
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
conversion of aminodeoxychorismate synthase into anthranilate synthase employing a bioinformatics method for predicting mutations required to functionally interconvert homologous enzymes. Complementation of an anthranilate synthase-deficient strain of Escherichia coli grown on minimal medium leads to several aminodeoxychorismate synthase mutants that allow growth in 6 days compared to 2 days for wild-type anthranilate synthase. The purified mutant enzymes catalyze the conversion of chorismate to anthranilate at rates that are about 50% of the rate of wild-type aminodeoxychorismate synthase-catalyzed conversion of chorismate to aminodeoxychorismate. The residues mutated do not contact the substrate
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