Information on EC 4.2.99.21 - isochorismate lyase and Organism(s) Pseudomonas aeruginosa

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Pseudomonas aeruginosa


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


The taxonomic range for the selected organisms is: Pseudomonas aeruginosa

EC NUMBER
COMMENTARY hide
4.2.99.21
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RECOMMENDED NAME
GeneOntology No.
isochorismate lyase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
isochorismate = salicylate + pyruvate
show the reaction diagram
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
salicylate biosynthesis I
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Biosynthesis of siderophore group nonribosomal peptides
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Biosynthesis of secondary metabolites
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Biosynthesis of antibiotics
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SYSTEMATIC NAME
IUBMB Comments
isochorismate pyruvate-lyase (salicylate-forming)
This enzyme is part of the pathway of salicylate formation from chorismate, and forms an integral part of pathways that produce salicylate-derived siderophores, such as pyochelin and yersiniabactin.
CAS REGISTRY NUMBER
COMMENTARY hide
383896-77-3
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
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PchB is a structural homologue of the AroQ chorismate mutases
metabolism
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the enzyme is involved in siderophore pyochelin via salicylate biosynthesis
physiological function
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the enzyme physiologically catalyzes the elimination of the enolpyruvyl side chain from isochorismate to make salicylate for incorporation into the siderophore pyochelin
additional information
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structure-function relationship, biocatalysis of pericyclic reactions, overview. For PchB, the pericyclic reaction is a concerted but asynchronous [1,5]-sigmatropic shift with a quantitative transfer of hydrogen from C2 to C9. Major structural difference between the apo form and the pyruvate-bound or the pyruvate-and salicylate-bound forms of PchB: the active site loop between helix 1 and helix 2 is disordered in the apo structure but fully ordered in the ligand-bound structures. The difference between the open and closed structures is due to a conserved active site lysine 42, which hydrogen bonds to a bound pyruvate molecule. Quantum mechanical/molecular mechanical molecular dynamics simulations, overview
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
isochorismate
salicylate + pyruvate
show the reaction diagram
additional information
?
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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
isochorismate
salicylate + pyruvate
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
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no cofactor required
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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no metal cofactor requirement
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
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not inhibitory at 1 mM: EDTA, EGTA, or o-phenanthroline. No substrate inhibition up to 1.2 mM
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00079 - 0.134
isochorismate
additional information
additional information
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Michaelis-Menten kinetics and thermodynamics, overview
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00038 - 1.76
isochorismate
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.017 - 1130
isochorismate
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
12.89
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pH 7.0, 37°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 9
active across the entire pH-range from 4 to 9, with a constant level of activity at pH 5 and above; activity range, pH profiles of recombinant wild-type and K42 mutant enzymes, overview
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
11500
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2 * 14000, SDS-PAGE, 2 * 11500, calculated
11563
x * 11563, calculated
14000
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2 * 14000, SDS-PAGE, 2 * 11500, calculated
31000 - 34000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 11563, calculated
dimer
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2 * 14000, SDS-PAGE, 2 * 11500, calculated
additional information
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enzyme is an intertwined dimer of three helices with connecting loops, and amino acids from each monomer participate in each of two active sites, crystallization data
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
apo-structure, to 2.35 A resolution, has one dimer per asymmetric unit with nitrate bound in an open active site. The loop between the first and second helices is disordered, providing a gateway for substrate entry and product exit. The pyruvate-bound structure, to 1.95 A resolution, has two dimers per asymmetric unit. One has two open active sites like the apo structure, and the other has two closed active sites with the loop between the first and second helices ordered for catalysis
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molecular dynamics simulations and averaged intermolecular substrate-protein distances, active-site volumes for reactants and transition state
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purified recombinant His-tagged wild-type enzyme and mutant K42E in complex with salicylate and pyruvate, hanging drop vapor diffusion method, mixing of 0.001 ml of 64 mg/ml wild-type protein with 0.001 ml of reservoir solution containing 0.2 M lithium sulfate, 0.1 M sodium acetate, pH 4.5, and 6% glycerol, mixing of 0.001 ml of 34 mg/ml mutant protein with 0.001 ml reservoir solution containing 0.004 M Gly-Gly, 0.100 M sodium acetate, pH 3.6, and 12% glycerol, ligands in 20fold molar excess, 25°C, 24-48 h, X-ray diffraction structure determination and analysis, modeling; wild-type and mutant K42E, to 1.95 and 1.79 A resolution, respectively, in complex with salicylate and pyruvate
X-ray crystallographic structures for mutant K42A with salicylate and pyruvate bound, to 2.5 A resolution, and for apo-I87T, to 2.15 A resolution. Circular dichroism studies of mutants K42A, K42Q, K42E, and K42H, A43P and I87T
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
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expression of a fusion of genes pchA and pchB from Pseudomonas aeruginosa, which encode isochorismate synthase and isochorismate pyruvate-lyase, in Arabidopsis thaliana
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
an entB mutant of Escherichia coli blocked in the conversion of isochorismate to 2,3-dihydro-2,3-dihydroxybenzoate forms salicylate when transformed with a pchB expression construct. Salicylate formation occurs in vitro when chorismate is incubated with a crude extract of Pseudomons aeruginosa containing overproduced PchA and PchB proteins, salicylate and pyruvate are formed in equimolar amounts
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A375T
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loss of the physiological isochorismate synthase catalytic efficiency by three orders of magnitude, and a 2fold gain in isochorismate-pyruvate lyase catalytic efficiency
A37I
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mutation increases the rate constant for the chorismate mutase activity by a factor of 1000, and also increases the isochorismate pyruvate lyase catalytic efficiency, by a factor of 6
A43P
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about 25% decrease in both chorismate mutase and isochorismate pyruvate lyase activity
C7A
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mutant has a reduced catalytic free energy of activation of up to 0.17 kcal/mol
C7A/K42C
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mutant has a reduced catalytic free energy of activation of up to 4.2 kcal/mol. Treatment with bromoethylamine leads to 64% recovery of activity
D310E
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physiological isochorismate synthase catalytic efficiency similar to wild-type, 3fold gain in isochorismate-pyruvate lyase catalytic efficiency
D310E/A375T
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similar activity in the isochorismate synthase and isochorismate-pyruvate lyase assays as the A375T variant
I87T
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structure demonstrates considerable mobility, decrease in both chorismate mutase and isochorismate pyruvate lyase activity
I88T
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no isochorismate pyruvate lyase activity, retains chorismate mutase activity
K42C
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mutant has a reduced catalytic free energy of activation of up to 4.4 kcal/mol. Treatment with bromoethylamine leads to 55% recovery of activity
K42Q
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almost complete loss of activity
Q91N
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20fold decrease in both isochorismate pyruvate lyase and chorismate mutase activity
R54K
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100fold decrease in both isochorismate pyruvate lyase and chorismate mutase activity
additional information
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a CM-deficient Escherichia coli mutant, which is auxotrophic for phenylalanine and tyrosine, is functionally complemented by the cloned pchB gene for growth in minimal medium
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
agriculture
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expression of a fusion of genes pchA and pchB from Pseudomonas aeruginosa, which encode isochorismate synthase and isochorismate pyruvate-lyase, in Arabidopsis thaliana, with targeting of the gene product either to the cytosol, c-SAS plants, or to the chloroplast, p-SAS plants. In p-SAS plants, the amount of free and conjugated SA is increased more than 20fold above wild type level. P-SAS plants show a strongly dwarfed phenotype and produce very few seeds. Targeting of SAS to the cytosol causes a slight increase in free salicylic acid and a significant threefold increase in conjugated salicylic acid. The modest increase in total salicylic content does not strongly induce the resistance marker PR-1, but results in enhanced disease resistance towards a virulent isolate of Peronospora parasitica. Increased resistance of c-SAS lines is paralleled with reduced seed production
biotechnology
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alternative computational rational approach to improve the secondary catalytic activity of enzymes, taking as a test case the IPL enzyme. The approach is based on the use of molecular dynamic simulations employing hybrid quantum mechanics/molecular mechanics methods that allow describing breaking and forming bonds
synthesis
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alternative computational rational approach to improve the secondary catalytic activity of enzymes, taking as a test case the IPL enzyme. The approach is based on the use of molecular dynamic simulations employing hybrid quantum mechanics/molecular mechanics methods that allow describing breaking and forming bonds