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Literature summary extracted from
- Subtle difference between benzene and toluene dioxygenases of Pseudomonas putida (2005), Appl. Environ. Microbiol., 71, 1570-1580.
Cloned(Commentary)
| EC Number | Cloned (Comment) | Organism |
|---|---|---|
| 1.14.12.3 | expressed in Escherichia coli JM109 | Pseudomonas putida |
| 1.14.12.3 | expression of wild-type and mutant enzymes in Escherichia coli strains JM109 and CJ236 | Pseudomonas putida |
| 1.14.12.11 | expressed in Escherichia coli JM109 | Pseudomonas putida |
| 1.14.12.11 | expressed in Escherichia coli strain JM109 | Pseudomonas putida |
Protein Variants
| EC Number | Protein Variants | Comment | Organism |
|---|---|---|---|
| 1.14.12.3 | A291S | reduced activity with ethylbenzene | Pseudomonas putida |
| 1.14.12.3 | E444D | no effect on activity | Pseudomonas putida |
| 1.14.12.3 | G404D | reduced activity with ethylbenzene | Pseudomonas putida |
| 1.14.12.3 | I301V | the mutation in the C-terminal part of subunit alpha enhances the substrate specificity for ethylbenzene, the mutant shows altered patterns of products formed from toluene and ethylbenzene, including monohydroxylated side chains | Pseudomonas putida |
| 1.14.12.3 | I301V/T305S/I307L/L309V | increased activity with ethylbenzene | Pseudomonas putida |
| 1.14.12.3 | I301V/T305S/I307L/L309V | the mutations in the C-terminal part of subunit alpha enhance the substrate specificity for ethylbenzene, the quadruple mutant also shows a high uncoupled rate of electron transfer without product formation | Pseudomonas putida |
| 1.14.12.3 | I307L | the mutation in the C-terminal part of subunit alpha enhances the substrate specificity for ethylbenzene, the mutant shows altered patterns of products formed from toluene and ethylbenzene, including monohydroxylated side chains | Pseudomonas putida |
| 1.14.12.3 | I412V | reduced activity with ethylbenzene | Pseudomonas putida |
| 1.14.12.3 | K436R | reduced activity with ethylbenzene | Pseudomonas putida |
| 1.14.12.3 | L285W | reduced activity with ethylbenzene | Pseudomonas putida |
| 1.14.12.3 | L285W/A291S/G404D | slightly reduced activity with ethylbenzene | Pseudomonas putida |
| 1.14.12.3 | L28W/A291S | reduced activity with ethylbenzene | Pseudomonas putida |
| 1.14.12.3 | L309V | the mutation in the C-terminal part of subunit alpha enhances the substrate specificity for ethylbenzene, the mutant shows altered patterns of products formed from toluene and ethylbenzene, including monohydroxylated side chains | Pseudomonas putida |
| 1.14.12.3 | additional information | construction of chimeric proteins and mutants of the benzene dioxygenase alpha subunit, the chimera are formed by benzene and toluene dioxygenases, the amino acid sequences of the alpha subunits of both enzymes differ at only 33 of 450 amino acids, these residues are primarily responsible for the change in specificity, the chimeric protein containing toluene dioxygenase C-terminal region residues 281 to 363 shows greater substrate preference for alkyl benzenes, identification of four amino acid substitutions in this region, I301V, T305S, I307L, and L309V, that particularly enhance the preference for ethylbenzene, structure modeling, overview | Pseudomonas putida |
| 1.14.12.3 | T305S | the mutation in the C-terminal part of subunit alpha enhances the substrate specificity for ethylbenzene, the mutant shows altered patterns of products formed from toluene and ethylbenzene, including monohydroxylated side chains | Pseudomonas putida |
| 1.14.12.3 | V324I/I327V | reduced activity with ethylbenzene | Pseudomonas putida |
Metals/Ions
| EC Number | Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|---|
| 1.14.12.3 | Fe2+ | the alpha subunit of the enzyme contains a catalytic [2Fe-2S] cluster involved in electron transfer, the catalytic nonheme iron center is also responsible for substrate specificity, overview | Pseudomonas putida |  |
| 1.14.12.11 | Fe2+ | contains a [2Fe-2S] cluster | Pseudomonas putida | |
Natural Substrates/ Products (Substrates)
| EC Number | Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 1.14.12.3 | benzene + NADH + O2 | Pseudomonas putida | bioremediation of aromatic environmental pollutants, initial step of benzene degradation | (1R,2S)-cis-cyclohexa-3,5-diene-1,2-diol + NAD+ | - |
? | |
| 1.14.12.11 | toluene + NADH + O2 | Pseudomonas putida | - |
(1S,2R)-3-methylcyclohexa-3,5-diene-1,2-diol + NAD+ | - |
? | |
Organism
| EC Number | Organism | UniProt | Comment | Textmining |
|---|---|---|---|---|
| 1.14.12.3 | Pseudomonas putida | - |
- |
- |
| 1.14.12.11 | Pseudomonas putida | - |
- |
- |
Reaction
| EC Number | Reaction | Comment | Organism | Reaction ID |
|---|---|---|---|---|
| 1.14.12.3 | benzene + NADH + H+ + O2 = cis-cyclohexa-3,5-diene-1,2-diol + NAD+ | the catalytic iron-sulfur proteins of the enzyme consist of two dissimilar alpha and beta subunits, the alpha subunit contains a [2Fe-2S] cluster involved in electron transfer, the catalytic nonheme iron center, and is also responsible for substrate specificity | Pseudomonas putida |
Specific Activity [micromol/min/mg]
| EC Number | Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|---|
| 1.14.12.3 | 0.025 | - |
with ethylbenzene as substrate | Pseudomonas putida |
| 1.14.12.3 | 0.071 | - |
with toluene as substrate | Pseudomonas putida |
| 1.14.12.3 | 0.115 | - |
with benzene as substrate | Pseudomonas putida |
| 1.14.12.11 | 0.045 | - |
with benzene as substrate | Pseudomonas putida |
| 1.14.12.11 | 0.054 | - |
with ethylbenzene as substrate | Pseudomonas putida |
| 1.14.12.11 | 0.056 | - |
with toluene as substrate | Pseudomonas putida |
Substrates and Products (Substrate)
| EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 1.14.12.3 | benzene + NADH + O2 | - |
Pseudomonas putida | (1R,2S)-cis-cyclohexa-3,5-diene-1,2-diol + NAD+ | - |
? | |
| 1.14.12.3 | benzene + NADH + O2 | bioremediation of aromatic environmental pollutants, initial step of benzene degradation | Pseudomonas putida | (1R,2S)-cis-cyclohexa-3,5-diene-1,2-diol + NAD+ | - |
? | |
| 1.14.12.3 | benzene + O2 + NADH | - |
Pseudomonas putida | cis-cyclohexa-3,5-diene-1,2-diol + NAD+ | - |
? | |
| 1.14.12.3 | ethylbenzene + NADH + O2 | - |
Pseudomonas putida | cis-2,3-dihydroxy-1-ethyl-cyclohexa-4,6-diene + 1-phenethyl alcohol + NAD+ | - |
? | |
| 1.14.12.3 | ethylbenzene + [reduced NADPH-hemoprotein reductase] + O2 | - |
Pseudomonas putida | cis-2,3-dihydroxy-1-ethyl-cyclohexa-4,6-diene + [oxidized NADPH-hemoprotein reductase] + H2O | - |
? | |
| 1.14.12.3 | toluene + NADH + O2 | - |
Pseudomonas putida | cis-2,3-dihydroxy-1-methyl-cyclohexa-4,6-diene + NAD+ | - |
? | |
| 1.14.12.11 | benzene + NADH + O2 | - |
Pseudomonas putida | benzene dihydrodiol + NAD+ | - |
? | |
| 1.14.12.11 | benzene + NADH + O2 | - |
Pseudomonas putida | benzene cis-dihydrodiol + NAD+ | - |
? | |
| 1.14.12.11 | ethylbenzene + NADH + H+ + O2 | - |
Pseudomonas putida | ethylbenzene cis-dihydrodiol + NAD+ | - |
? | |
| 1.14.12.11 | ethylbenzene + NADH + O2 | - |
Pseudomonas putida | ? | - |
? | |
| 1.14.12.11 | toluene + NADH + O2 | - |
Pseudomonas putida | (1S,2R)-3-methylcyclohexa-3,5-diene-1,2-diol + NAD+ | - |
? | |
Subunits
| EC Number | Subunits | Comment | Organism |
|---|---|---|---|
| 1.14.12.3 | dimer | alpha, beta, iron sulfur protein component | Pseudomonas putida |
| 1.14.12.3 | dimer | the enzyme consist of two dissimilar alpha and beta subunits, alpha subunit structure modeling | Pseudomonas putida |
Synonyms
| EC Number | Synonyms | Comment | Organism |
|---|---|---|---|
| 1.14.12.3 | benzene dioxygenase | - |
Pseudomonas putida |
| 1.14.12.3 | More | enzyme system consisting of three components: a flavoprotein reductase, a ferredoxin and a catalytic iron sulfur protein | Pseudomonas putida |
| 1.14.12.11 | ISPTOD | - |
Pseudomonas putida |
Temperature Optimum [°C]
| EC Number | Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|---|
| 1.14.12.3 | 22 | - |
assay at room temperature | Pseudomonas putida |
pH Optimum
| EC Number | pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|---|
| 1.14.12.3 | 7.2 | - |
assay at | Pseudomonas putida |
Cofactor
| EC Number | Cofactor | Comment | Organism | Structure |
|---|---|---|---|---|
| 1.14.12.3 | Ferredoxin | - |
Pseudomonas putida | |
| 1.14.12.3 | iron-sulfur centre | [2Fe-2S] cluster | Pseudomonas putida | |
| 1.14.12.3 | NADH | - |
Pseudomonas putida | |
| 1.14.12.11 | NADH | - |
Pseudomonas putida | |
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