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Literature summary extracted from

  • Koetsier, M.J.; Jekel, P.A.; van den Berg, M.A.; Bovenberg, R.A.; Janssen, D.B.
    Characterization of a phenylacetate-CoA ligase from Penicillium chrysogenum (2008), Biochem. J., 417, 467-476.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
6.2.1.2 gene phl, sequence analysis, functional expression as C-terminally His6-tagged maltose binding protein fusion in Escherichia coli, expression of His-tagged mutants Penicillium chrysogenum
6.2.1.30 gene phl, sequence analysis, functional expression as C-terminally His6-tagged maltose binding protein fusion in Escherichia coli, expression of His-tagged mutants Penicillium chrysogenum

Protein Variants

EC Number Protein Variants Comment Organism
6.2.1.2 F307A site-directed mutagenesis, the mutant shows an increased Km for phenoxyacetic acid compared to the wild-type enzyme Penicillium chrysogenum
6.2.1.2 F335A site-directed mutagenesis, the mutant shows an increased Km for phenoxyacetic acid compared to the wild-type enzyme Penicillium chrysogenum
6.2.1.2 additional information construction of mutants with substrate binding pocket residues exchanged for alanine, the mutants show altered kinetics, overview Penicillium chrysogenum
6.2.1.2 V270A site-directed mutagenesis, the mutant activity is similar to the wild-type enzyme Penicillium chrysogenum
6.2.1.2 V370A site-directed mutagenesis, the mutant activity is similar to the wild-type enzyme Penicillium chrysogenum
6.2.1.2 Y267A site-directed mutagenesis, the mutant shows an increased Km for phenoxyacetic acid compared to the wild-type enzyme Penicillium chrysogenum
6.2.1.30 F307A site-directed mutagenesis, the mutant shows an increased Km for phenoxyacetic acid compared to the wild-type enzyme Penicillium chrysogenum
6.2.1.30 F335A site-directed mutagenesis, the mutant shows an increased Km for phenoxyacetic acid compared to the wild-type enzyme Penicillium chrysogenum
6.2.1.30 additional information construction of mutants with substrate binding pocket residues exchanged for alanine, the mutants show altered kinetics, overview Penicillium chrysogenum
6.2.1.30 V270A site-directed mutagenesis, the mutant activity is similar to the wild-type enzyme Penicillium chrysogenum
6.2.1.30 V370A site-directed mutagenesis, the mutant activity is similar to the wild-type enzyme Penicillium chrysogenum
6.2.1.30 Y267A site-directed mutagenesis, the mutant shows an increased Km for phenoxyacetic acid compared to the wild-type enzyme Penicillium chrysogenum

General Stability

EC Number General Stability Organism
6.2.1.2 presence of 200 mM NaCl is essential for the stability of the enzyme and storage or conversions in buffer lacking NaCl resulted in rapid loss of activity Penicillium chrysogenum
6.2.1.30 presence of 200 mM NaCl is essential for the stability of the enzyme and storage or conversions in buffer lacking NaCl resulted in rapid loss of activity Penicillium chrysogenum

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
6.2.1.2 additional information
-
additional information kinetics with diverse substrates, overview Penicillium chrysogenum
6.2.1.2 0.001
-
myristic acid below, pH 8.5, 30°C, recombinant enzyme Penicillium chrysogenum
6.2.1.2 0.12
-
CoA pH 8.5, 30°C, recombinant enzyme, with phenylacetic acid Penicillium chrysogenum
6.2.1.2 0.19
-
trans-cinnamic acid pH 8.5, 30°C, recombinant enzyme Penicillium chrysogenum
6.2.1.2 0.48
-
ATP pH 8.5, 30°C, recombinant enzyme, with trans-cinnamic acid Penicillium chrysogenum
6.2.1.2 0.65
-
ATP pH 8.5, 30°C, recombinant enzyme, with phenylacetic acid Penicillium chrysogenum
6.2.1.2 0.94
-
CoA pH 8.5, 30°C, recombinant enzyme, with trans-cinnamic acid Penicillium chrysogenum
6.2.1.2 2.1
-
Phenoxyacetate pH 8.5, 30°C, recombinant enzyme Penicillium chrysogenum
6.2.1.2 5.2 6.1 phenylacetate pH 8.5, 30°C, recombinant enzyme Penicillium chrysogenum
6.2.1.2 80
-
propionic acid pH 8.5, 30°C, recombinant enzyme Penicillium chrysogenum
6.2.1.30 additional information
-
additional information kinetics of wild-type and mutant enzymes with diverse substrates, overview Penicillium chrysogenum
6.2.1.30 0.001
-
myristic acid below, pH 8.5, 30°C, recombinant enzyme Penicillium chrysogenum
6.2.1.30 0.12
-
CoA pH 8.5, 30°C, recombinant enzyme, with phenylacetic acid Penicillium chrysogenum
6.2.1.30 0.19
-
trans-cinnamic acid pH 8.5, 30°C, recombinant enzyme Penicillium chrysogenum
6.2.1.30 0.48
-
ATP pH 8.5, 30°C, recombinant enzyme, with trans-cinnamic acid Penicillium chrysogenum
6.2.1.30 0.65
-
ATP pH 8.5, 30°C, recombinant enzyme, with phenylacetic acid Penicillium chrysogenum
6.2.1.30 0.94
-
CoA pH 8.5, 30°C, recombinant enzyme, with trans-cinnamic acid Penicillium chrysogenum
6.2.1.30 2.1
-
Phenoxyacetate pH 8.5, 30°C, recombinant enzyme Penicillium chrysogenum
6.2.1.30 5.2 6.1 phenylacetate pH 8.5, 30°C, recombinant enzyme Penicillium chrysogenum
6.2.1.30 24.4
-
Butyric acid pH 8.5, 30°C, recombinant enzyme Penicillium chrysogenum
6.2.1.30 80
-
propionic acid pH 8.5, 30°C, recombinant enzyme Penicillium chrysogenum

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
6.2.1.2 Mg2+ dependent on, optimal at 5 mM Penicillium chrysogenum
6.2.1.2 NaCl stabilizes at 200 mM Penicillium chrysogenum
6.2.1.30 Mg2+ dependent on, optimal at 5 mM Penicillium chrysogenum
6.2.1.30 NaCl stabilizes at 200 mM Penicillium chrysogenum

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
6.2.1.2 62600
-
x * 62600, recombinant enzyme, SDS-PAGE Penicillium chrysogenum
6.2.1.30 62600
-
x * 62600, recombinant enzyme, SDS-PAGE Penicillium chrysogenum

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
6.2.1.2 ATP + phenylacetate + CoA Penicillium chrysogenum enzymatic activation of phenylacetic acid to phenylacetyl-CoA is an important step in the biosynthesis of the beta-lactam antibiotic penicillin G by the fungus Penicillium chrysogenum, CoA esters of PAA and phenoxyacetic acid act as acyl donor in the exchange of the aminoadipyl side chain of isopenicillin N to produce penicillin G or penicillin V AMP + diphosphate + phenylacetyl-CoA
-
?
6.2.1.30 ATP + butyric acid + CoA Penicillium chrysogenum
-
AMP + diphosphate + butyryl-CoA
-
?
6.2.1.30 ATP + phenylacetate + CoA Penicillium chrysogenum enzymatic activation of phenylacetic acid to phenylacetyl-CoA is an important step in the biosynthesis of the beta-lactam antibiotic penicillin G by the fungus Penicillium chrysogenum, CoA esters of phenylacetic acid and phenoxyacetic acid act as acyl donor in the exchange of the aminoadipyl side chain of isopenicillin N to produce penicillin G or penicillin V AMP + diphosphate + phenylacetyl-CoA
-
?

Organism

EC Number Organism UniProt Comment Textmining
6.2.1.2 Penicillium chrysogenum O74725 gene phl or pclA; gene phl
-
6.2.1.30 Penicillium chrysogenum O74725 gene phl or pclA; gene phl
-

Purification (Commentary)

EC Number Purification (Comment) Organism
6.2.1.2 recombinant C-terminally His6-tagged maltose binding protein fusion from Escherichia coli by nickel affinity chromatography, the MBP is cleaved off by factor Xa, imidazole is eliminated by gel filtration, recombinant His-tagged mutants Penicillium chrysogenum
6.2.1.30 recombinant C-terminally His6-tagged maltose binding protein fusion from Escherichia coli by nickel affinity chromatography, the MBP is cleaved off by factor Xa, imidazole is eliminated by gel filtration, recombinant His-tagged mutants Penicillium chrysogenum

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
6.2.1.2 additional information
-
-
Penicillium chrysogenum
6.2.1.2 0.02
-
purified recombinant enzyme Penicillium chrysogenum
6.2.1.30 additional information
-
-
Penicillium chrysogenum
6.2.1.30 0.02
-
purified recombinant enzyme Penicillium chrysogenum

Storage Stability

EC Number Storage Stability Organism
6.2.1.2 -20°C, purified recobnant protein in TANG buffer, several months without significant loss of activity Penicillium chrysogenum
6.2.1.30 -20°C, purified recombinant protein in TANG buffer, several months without significant loss of activity Penicillium chrysogenum

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
6.2.1.2 ATP + 3,4-methylenedioxycinnamic acid + CoA high activity Penicillium chrysogenum AMP + diphosphate + 3,4-methylenedioxy-cinnamoyl-CoA
-
?
6.2.1.2 ATP + 3-ethoxycinnamic acid + CoA high activity Penicillium chrysogenum AMP + diphosphate + 3-ethoxy-cinnamoyl-CoA
-
?
6.2.1.2 ATP + 3-methoxycinnamic acid + CoA high activity Penicillium chrysogenum AMP + diphosphate + 3-methoxy-cinnamoyl-CoA
-
?
6.2.1.2 ATP + caproic acid + CoA
-
Penicillium chrysogenum AMP + diphosphate + caproyl-CoA
-
?
6.2.1.2 ATP + myristic acid + CoA
-
Penicillium chrysogenum AMP + diphosphate + myristoyl-CoA
-
?
6.2.1.2 ATP + phenoxyacetate + CoA i.e. POA, low activity Penicillium chrysogenum AMP + diphosphate + phenoxyacetyl-CoA
-
?
6.2.1.2 ATP + phenylacetate + CoA enzymatic activation of phenylacetic acid to phenylacetyl-CoA is an important step in the biosynthesis of the beta-lactam antibiotic penicillin G by the fungus Penicillium chrysogenum, CoA esters of PAA and phenoxyacetic acid act as acyl donor in the exchange of the aminoadipyl side chain of isopenicillin N to produce penicillin G or penicillin V Penicillium chrysogenum AMP + diphosphate + phenylacetyl-CoA
-
?
6.2.1.2 ATP + phenylacetate + CoA i.e. PAA Penicillium chrysogenum AMP + diphosphate + phenylacetyl-CoA
-
?
6.2.1.2 ATP + propionic acid + CoA low activity Penicillium chrysogenum AMP + diphosphate + propionyl-CoA
-
?
6.2.1.2 ATP + trans-4-coumaric acid + CoA
-
Penicillium chrysogenum AMP + diphosphate + trans-4-coumaroyl-CoA
-
?
6.2.1.2 ATP + trans-cinnamic acid + CoA 1000fold higher activity compared to PAA Penicillium chrysogenum AMP + diphosphate + trans-cinnamoyl-CoA
-
?
6.2.1.2 additional information the enzyme also belongs to EC 6.2.1.30, substrate specificity, the more substituted compounds ferulic acid, caffeic acid and sinapic acid, which are substrates for most 4-coumarate CoA ligases, are very poor substrates for the enzyme. With the exception of acetic acid, all short and medium chain fatty acids tested are converted by the enzyme, the enzyme is able to activate all the side chains of these naturally occurring lactam side products, overview. Residues H265, I266, Y267, V270, F307, F335, G337, A338, G361, T369, V370, and K557 are involved in substrate binding Penicillium chrysogenum ?
-
?
6.2.1.30 ATP + 3,4-methylenedioxycinnamic acid + CoA high activity Penicillium chrysogenum AMP + diphosphate + 3,4-methylenedioxycinnamoyl-CoA
-
?
6.2.1.30 ATP + 3-ethoxycinnamic acid + CoA high activity Penicillium chrysogenum AMP + diphosphate + 3-ethoxy-cinnamoyl-CoA
-
?
6.2.1.30 ATP + 3-methoxycinnamic acid + CoA high activity Penicillium chrysogenum AMP + diphosphate + 3-methoxy-cinnamoyl-CoA
-
?
6.2.1.30 ATP + butyric acid + CoA
-
Penicillium chrysogenum AMP + diphosphate + butyryl-CoA
-
?
6.2.1.30 ATP + caproic acid + CoA
-
Penicillium chrysogenum AMP + diphosphate + caproyl-CoA
-
?
6.2.1.30 ATP + myristic acid + CoA
-
Penicillium chrysogenum AMP + diphosphate + myristoyl-CoA
-
?
6.2.1.30 ATP + phenoxyacetate + CoA low activity Penicillium chrysogenum AMP + diphosphate + phenoxyacetyl-CoA
-
?
6.2.1.30 ATP + phenylacetate + CoA
-
Penicillium chrysogenum AMP + diphosphate + phenylacetyl-CoA
-
?
6.2.1.30 ATP + phenylacetate + CoA enzymatic activation of phenylacetic acid to phenylacetyl-CoA is an important step in the biosynthesis of the beta-lactam antibiotic penicillin G by the fungus Penicillium chrysogenum, CoA esters of phenylacetic acid and phenoxyacetic acid act as acyl donor in the exchange of the aminoadipyl side chain of isopenicillin N to produce penicillin G or penicillin V Penicillium chrysogenum AMP + diphosphate + phenylacetyl-CoA
-
?
6.2.1.30 ATP + propionic acid + CoA low activity Penicillium chrysogenum AMP + diphosphate + propionyl-CoA
-
?
6.2.1.30 ATP + trans-4-coumaric acid + CoA
-
Penicillium chrysogenum AMP + diphosphate + trans-4-coumaroyl-CoA
-
?
6.2.1.30 ATP + trans-cinnamic acid + CoA 1000fold higher activity compared to PAA Penicillium chrysogenum AMP + diphosphate + trans-cinnamoyl-CoA
-
?
6.2.1.30 additional information the enzyme also belongs to EC 6.2.1.2, substrate specificity of PCL, the more substituted compounds ferulic acid, caffeic acid and sinapic acid, which are substrates for most 4-coumarate CoA ligases, are very poor substrates for PCL. With the exception of acetic acid, all short and medium chain fatty acids tested are converted by PCL, PCL is able to activate all the side chains of these naturally occurring lactam side products, overview. Residues H265, I266, Y267, V270, F307, F335, G337, A338, G361, T369, V370, and K557 are involved in substrate binding Penicillium chrysogenum ?
-
?

Subunits

EC Number Subunits Comment Organism
6.2.1.2 ? x * 62600, recombinant enzyme, SDS-PAGE Penicillium chrysogenum
6.2.1.2 More enzyme structure modelling of wild-type and mutant enzymes using the crystal structure of the luciferase from firefly species Luciola cruciata as template, PDB ID 2D1S Penicillium chrysogenum
6.2.1.30 ? x * 62600, recombinant enzyme, SDS-PAGE Penicillium chrysogenum
6.2.1.30 More enzyme structure modelling of wild-type and mutant enzymes using the crystal structure of the luciferase from firefly species Luciola cruciata as template, PDB ID 2D1S Penicillium chrysogenum

Synonyms

EC Number Synonyms Comment Organism
6.2.1.2 More cf. 6.2.1.30 Penicillium chrysogenum
6.2.1.30 More cf. 6.2.1.2 Penicillium chrysogenum
6.2.1.30 PCL
-
Penicillium chrysogenum

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
6.2.1.2 35 40
-
Penicillium chrysogenum
6.2.1.30 35 40
-
Penicillium chrysogenum

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
6.2.1.2 1.3 1.9 phenylacetate pH 8.5, 30°C, recombinant enzyme Penicillium chrysogenum
6.2.1.2 1.5
-
CoA pH 8.5, 30°C, recombinant enzyme, with phenylacetic acid Penicillium chrysogenum
6.2.1.2 1.7
-
ATP pH 8.5, 30°C, recombinant enzyme, with phenylacetic acid Penicillium chrysogenum
6.2.1.2 12
-
Phenoxyacetate pH 8.5, 30°C, recombinant enzyme Penicillium chrysogenum
6.2.1.2 41.2
-
trans-cinnamic acid pH 8.5, 30°C, recombinant enzyme Penicillium chrysogenum
6.2.1.2 42.9
-
ATP pH 8.5, 30°C, recombinant enzyme, with trans-cinnamic acid Penicillium chrysogenum
6.2.1.2 60.5
-
CoA pH 8.5, 30°C, recombinant enzyme, with trans-cinnamic acid Penicillium chrysogenum
6.2.1.30 1.3 1.9 phenylacetate pH 8.5, 30°C, recombinant enzyme Penicillium chrysogenum
6.2.1.30 1.5
-
CoA pH 8.5, 30°C, recombinant enzyme, with phenylacetic acid Penicillium chrysogenum
6.2.1.30 1.7
-
ATP pH 8.5, 30°C, recombinant enzyme, with phenylacetic acid Penicillium chrysogenum
6.2.1.30 9.7
-
Butyric acid pH 8.5, 30°C, recombinant enzyme Penicillium chrysogenum
6.2.1.30 12
-
Phenoxyacetate pH 8.5, 30°C, recombinant enzyme Penicillium chrysogenum
6.2.1.30 41.2
-
trans-cinnamic acid pH 8.5, 30°C, recombinant enzyme Penicillium chrysogenum
6.2.1.30 42.9
-
ATP pH 8.5, 30°C, recombinant enzyme, with trans-cinnamic acid Penicillium chrysogenum
6.2.1.30 60.5
-
CoA pH 8.5, 30°C, recombinant enzyme, with trans-cinnamic acid Penicillium chrysogenum

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
6.2.1.2 8.5
-
-
Penicillium chrysogenum
6.2.1.30 8.5
-
-
Penicillium chrysogenum

Cofactor

EC Number Cofactor Comment Organism Structure
6.2.1.2 ATP dependent on Penicillium chrysogenum
6.2.1.30 ATP dependent on Penicillium chrysogenum

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
6.2.1.2 additional information
-
additional information inhibition kinetics Penicillium chrysogenum
6.2.1.30 additional information
-
additional information inhibition kinetics Penicillium chrysogenum