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Literature summary for 6.2.1.2 extracted from
- Characterization of a phenylacetate-CoA ligase from Penicillium chrysogenum (2008), Biochem. J., 417, 467-476.
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| 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
| Protein Variants | Comment | Organism |
|---|---|---|
| F307A | site-directed mutagenesis, the mutant shows an increased Km for phenoxyacetic acid compared to the wild-type enzyme | Penicillium chrysogenum |
| F335A | site-directed mutagenesis, the mutant shows an increased Km for phenoxyacetic acid compared to the wild-type enzyme | Penicillium chrysogenum |
| additional information | construction of mutants with substrate binding pocket residues exchanged for alanine, the mutants show altered kinetics, overview | Penicillium chrysogenum |
| V270A | site-directed mutagenesis, the mutant activity is similar to the wild-type enzyme | Penicillium chrysogenum |
| V370A | site-directed mutagenesis, the mutant activity is similar to the wild-type enzyme | Penicillium chrysogenum |
| Y267A | site-directed mutagenesis, the mutant shows an increased Km for phenoxyacetic acid compared to the wild-type enzyme | Penicillium chrysogenum |
General Stability
| General Stability | Organism |
|---|---|
| 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]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | kinetics with diverse substrates, overview | Penicillium chrysogenum | |
| 0.001 | - |
myristic acid | below, pH 8.5, 30°C, recombinant enzyme | Penicillium chrysogenum |  |
| 0.12 | - |
CoA | pH 8.5, 30°C, recombinant enzyme, with phenylacetic acid | Penicillium chrysogenum | |
| 0.19 | - |
trans-cinnamic acid | pH 8.5, 30°C, recombinant enzyme | Penicillium chrysogenum | |
| 0.48 | - |
ATP | pH 8.5, 30°C, recombinant enzyme, with trans-cinnamic acid | Penicillium chrysogenum | |
| 0.65 | - |
ATP | pH 8.5, 30°C, recombinant enzyme, with phenylacetic acid | Penicillium chrysogenum | |
| 0.94 | - |
CoA | pH 8.5, 30°C, recombinant enzyme, with trans-cinnamic acid | Penicillium chrysogenum | |
| 2.1 | - |
Phenoxyacetate | pH 8.5, 30°C, recombinant enzyme | Penicillium chrysogenum | |
| 5.2 | 6.1 | phenylacetate | pH 8.5, 30°C, recombinant enzyme | Penicillium chrysogenum | |
| 80 | - |
propionic acid | pH 8.5, 30°C, recombinant enzyme | Penicillium chrysogenum | |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Mg2+ | dependent on, optimal at 5 mM | Penicillium chrysogenum | |
| NaCl | stabilizes at 200 mM | Penicillium chrysogenum | |
Molecular Weight [Da]
| Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
|---|---|---|---|
| 62600 | - |
x * 62600, recombinant enzyme, SDS-PAGE | Penicillium chrysogenum |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 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 | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Penicillium chrysogenum | O74725 | gene phl or pclA; gene phl | - |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| 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]
| Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|
| additional information | - |
- |
Penicillium chrysogenum |
| 0.02 | - |
purified recombinant enzyme | Penicillium chrysogenum |
Storage Stability
| Storage Stability | Organism |
|---|---|
| -20°C, purified recobnant protein in TANG buffer, several months without significant loss of activity | Penicillium chrysogenum |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| ATP + 3,4-methylenedioxycinnamic acid + CoA | high activity | Penicillium chrysogenum | AMP + diphosphate + 3,4-methylenedioxy-cinnamoyl-CoA | - |
? | |
| ATP + 3-ethoxycinnamic acid + CoA | high activity | Penicillium chrysogenum | AMP + diphosphate + 3-ethoxy-cinnamoyl-CoA | - |
? | |
| ATP + 3-methoxycinnamic acid + CoA | high activity | Penicillium chrysogenum | AMP + diphosphate + 3-methoxy-cinnamoyl-CoA | - |
? | |
| ATP + caproic acid + CoA | - |
Penicillium chrysogenum | AMP + diphosphate + caproyl-CoA | - |
? | |
| ATP + myristic acid + CoA | - |
Penicillium chrysogenum | AMP + diphosphate + myristoyl-CoA | - |
? | |
| ATP + phenoxyacetate + CoA | i.e. POA, low activity | Penicillium chrysogenum | AMP + diphosphate + phenoxyacetyl-CoA | - |
? | |
| 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 | - |
? | |
| ATP + phenylacetate + CoA | i.e. PAA | Penicillium chrysogenum | AMP + diphosphate + phenylacetyl-CoA | - |
? | |
| ATP + propionic acid + CoA | low activity | Penicillium chrysogenum | AMP + diphosphate + propionyl-CoA | - |
? | |
| ATP + trans-4-coumaric acid + CoA | - |
Penicillium chrysogenum | AMP + diphosphate + trans-4-coumaroyl-CoA | - |
? | |
| ATP + trans-cinnamic acid + CoA | 1000fold higher activity compared to PAA | Penicillium chrysogenum | AMP + diphosphate + trans-cinnamoyl-CoA | - |
? | |
| 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 | ? | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| ? | x * 62600, recombinant enzyme, SDS-PAGE | Penicillium chrysogenum |
| 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
| Synonyms | Comment | Organism |
|---|---|---|
| More | cf. 6.2.1.30 | Penicillium chrysogenum |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 35 | 40 | - |
Penicillium chrysogenum |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 1.3 | 1.9 | phenylacetate | pH 8.5, 30°C, recombinant enzyme | Penicillium chrysogenum | |
| 1.5 | - |
CoA | pH 8.5, 30°C, recombinant enzyme, with phenylacetic acid | Penicillium chrysogenum | |
| 1.7 | - |
ATP | pH 8.5, 30°C, recombinant enzyme, with phenylacetic acid | Penicillium chrysogenum | |
| 12 | - |
Phenoxyacetate | pH 8.5, 30°C, recombinant enzyme | Penicillium chrysogenum | |
| 41.2 | - |
trans-cinnamic acid | pH 8.5, 30°C, recombinant enzyme | Penicillium chrysogenum | |
| 42.9 | - |
ATP | pH 8.5, 30°C, recombinant enzyme, with trans-cinnamic acid | Penicillium chrysogenum | |
| 60.5 | - |
CoA | pH 8.5, 30°C, recombinant enzyme, with trans-cinnamic acid | Penicillium chrysogenum | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 8.5 | - |
- |
Penicillium chrysogenum |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| ATP | dependent on | Penicillium chrysogenum | |
Ki Value [mM]
| Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | inhibition kinetics | Penicillium chrysogenum |
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