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Literature summary for 1.14.12.3 extracted from

  • Bagneris, C.; Cammack, R.; Mason, J.R.
    Subtle difference between benzene and toluene dioxygenases of Pseudomonas putida (2005), Appl. Environ. Microbiol., 71, 1570-1580.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli JM109 Pseudomonas putida
expression of wild-type and mutant enzymes in Escherichia coli strains JM109 and CJ236 Pseudomonas putida

Protein Variants

Protein Variants Comment Organism
A291S reduced activity with ethylbenzene Pseudomonas putida
E444D no effect on activity Pseudomonas putida
G404D reduced activity with ethylbenzene Pseudomonas putida
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
I301V/T305S/I307L/L309V increased activity with ethylbenzene Pseudomonas putida
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
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
I412V reduced activity with ethylbenzene Pseudomonas putida
K436R reduced activity with ethylbenzene Pseudomonas putida
L285W reduced activity with ethylbenzene Pseudomonas putida
L285W/A291S/G404D slightly reduced activity with ethylbenzene Pseudomonas putida
L28W/A291S reduced activity with ethylbenzene Pseudomonas putida
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
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
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
V324I/I327V reduced activity with ethylbenzene Pseudomonas putida

Metals/Ions

Metals/Ions Comment Organism Structure
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

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
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+
-
?

Organism

Organism UniProt Comment Textmining
Pseudomonas putida
-
-
-

Reaction

Reaction Comment Organism Reaction ID
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]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
0.025
-
with ethylbenzene as substrate Pseudomonas putida
0.071
-
with toluene as substrate Pseudomonas putida
0.115
-
with benzene as substrate Pseudomonas putida

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
benzene + NADH + O2
-
Pseudomonas putida (1R,2S)-cis-cyclohexa-3,5-diene-1,2-diol + NAD+
-
?
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+
-
?
benzene + O2 + NADH
-
Pseudomonas putida cis-cyclohexa-3,5-diene-1,2-diol + NAD+
-
?
ethylbenzene + NADH + O2
-
Pseudomonas putida cis-2,3-dihydroxy-1-ethyl-cyclohexa-4,6-diene + 1-phenethyl alcohol + NAD+
-
?
ethylbenzene + [reduced NADPH-hemoprotein reductase] + O2
-
Pseudomonas putida cis-2,3-dihydroxy-1-ethyl-cyclohexa-4,6-diene + [oxidized NADPH-hemoprotein reductase] + H2O
-
?
toluene + NADH + O2
-
Pseudomonas putida cis-2,3-dihydroxy-1-methyl-cyclohexa-4,6-diene + NAD+
-
?

Subunits

Subunits Comment Organism
dimer alpha, beta, iron sulfur protein component Pseudomonas putida
dimer the enzyme consist of two dissimilar alpha and beta subunits, alpha subunit structure modeling Pseudomonas putida

Synonyms

Synonyms Comment Organism
benzene dioxygenase
-
Pseudomonas putida
More enzyme system consisting of three components: a flavoprotein reductase, a ferredoxin and a catalytic iron sulfur protein Pseudomonas putida

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
22
-
assay at room temperature Pseudomonas putida

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.2
-
assay at Pseudomonas putida

Cofactor

Cofactor Comment Organism Structure
Ferredoxin
-
Pseudomonas putida
iron-sulfur centre [2Fe-2S] cluster Pseudomonas putida
NADH
-
Pseudomonas putida