Information on EC 1.14.13.236 - toluene 4-monooxygenase

for references in articles please use BRENDA:EC1.14.13.236
Word Map on EC 1.14.13.236
Please wait a moment until all data is loaded. This message will disappear when all data is loaded.
Specify your search results
Select one or more organisms in this record:

The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.14.13.236
-
RECOMMENDED NAME
GeneOntology No.
toluene 4-monooxygenase
-
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
toluene + NADH + H+ + O2 = 4-methylphenol + NAD+ + H2O
show the reaction diagram
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
toluene degradation to 2-oxopent-4-enoate (via 4-methylcatechol)
-
-
toluene degradation to 4-methylphenol
-
-
Toluene degradation
-
-
Metabolic pathways
-
-
Microbial metabolism in diverse environments
-
-
SYSTEMATIC NAME
IUBMB Comments
toluene,NADH:oxygen oxidoreductase (4-hydroxylating)
This bacterial enzyme belongs to a family of soluble diiron hydroxylases that includes toluene-, benzene-, xylene- and methane monooxygenases, phenol hydroxylases, and alkene epoxidases. The enzyme comprises a four-component complex that includes a hydroxylase, NADH-ferredoxin oxidoreductase, a Rieske-type [2Fe-2S] ferredoxin, and an effector protein.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
A0A0D3QM77 i.e. subunit TmoA, A0A0D3QME2 i.e. subunit TmoB, A0A0D3QLU4 i.e. subunit TmoC, A0A0D3QM47 i.e. subunit TmD, A0A0D3QMJ7 i.e. subunit TmoE, A0A0D3QM80 i.e. subunit TmoF
A0A0D3QM77 and A0A0D3QME2 and A0A0D3QLU4 and A0A0D3QM47 and A0A0D3QMJ7 and A0A0D3QM80
UniProt
Manually annotated by BRENDA team
isolated from a tar oil-contaminated site
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
1,1-diethylcyclopropane + NADH + H+ + O2
? + NAD+ + H2O
show the reaction diagram
1,1-dimethylcyclopropane + NADH + H+ + O2
? + NAD+ + H2O
show the reaction diagram
2 indole + 3 NADH + 3 H+ + 3 O2
indirubin + 3 NAD+ + 3 H2O
show the reaction diagram
A0A0D3QM77 and A0A0D3QME2 and A0A0D3QLU4 and A0A0D3QM47 and A0A0D3QMJ7 and A0A0D3QM80
-
reaction via C-3 oxidation to indoxyl, oxidation to isatin, and recombinantion of isatin and indoxyl to indirubin
-
?
2-phenylethanol + 2 NADH + 2 H+ + O2
3,4-dihydroxyphenylethanol + 2 NAD+ + 2 H2O
show the reaction diagram
2-phenylethanol + NADH + H+ + O2
m-tyrosol + p-tyrosol + NAD+ + H2O
show the reaction diagram
2-phenylethanol + NADH + H+ + O2
o-tyrosol + m-tyrosol + NAD+ + H2O
show the reaction diagram
2-xylene + NADH + H+ + O2
3,4-dimethylphenol + NAD+ + H2O
show the reaction diagram
3-xylene + NADH + H+ + O2
2,4-dimethylphenol + NAD+ + H2O
show the reaction diagram
4-xylene + NADH + H+ + O2
2,5-dimethylphenol + 4-methyl benzyl alcohol + NAD+ + H2O
show the reaction diagram
anisole + NADH + H+ + O2
4-methoxyphenol + NAD+ + H2O
show the reaction diagram
benzene + NADH + H+ + O2
phenol + NAD+ + H2O
show the reaction diagram
chlorobenzene + NADH + H+ + O2
4-chlorophenol + NAD+ + H2O
show the reaction diagram
fluorobenzene + NADH + H+ + O2
4-fluorophenol 4-fluorocatechol + NAD+ + H2O
show the reaction diagram
m-cresol + NADH + H+ + O2
4-methylcatechol + NAD+ + H2O
show the reaction diagram
m-tyrosol + 2 NADH + 2 H+ + O2
2,3-dihydroxyphenylethanol + 2 NAD+ + 2 H2O
show the reaction diagram
m-tyrosol + 2 NADH + 2 H+ + O2
3,4-dihydroxyphenylethanol + 2 NAD+ + 2 H2O
show the reaction diagram
methoxybenzene + NADH + H+ + O2
4-methoxyphenol + NAD+ + H2O
show the reaction diagram
-
more than 99% 4-methoxyphenol
-
?
methyl 4-tolyl sulfide + NADH + H+ + O2
methyl 4-tolyl sulfoxide + NAD+ + H2O
show the reaction diagram
methyl p-tolyl sulfide + NADH + H+ + O2
methyl p-tolyl sulfoxide + NAD+ + H2O
show the reaction diagram
methyl phenyl sulfide + NADH + H+ + O2
methyl phenyl sulfoxide + NAD+ + H2O
show the reaction diagram
naphthalene + NADH + H+ + O2
2-naphthol + NAD+ + H2O
show the reaction diagram
nitrobenzene + NADH + H+ + O2
4-nitrophenol + NAD+ + H2O
show the reaction diagram
norcarane + NADH + H+ + O2
endo-2-norcaranol + exo-2-norcaranol + endo-3-norcaranol + NAD+ + H2O
show the reaction diagram
o-cresol + NADH + H+ + O2
3-methylcatechol + NAD+ + H2O
show the reaction diagram
o-methoxyphenol + NADH + H+ + O2
4-methoxyresorcinol + 3-methoxycatechol + NAD+ + H2O
show the reaction diagram
o-tyrosol + 2 NADH + 2 H+ + O2
2,3-dihydroxyphenylethanol + 2 NAD+ + 2 H2O
show the reaction diagram
o-tyrosol + NADH + H+ + O2
2,3-dihydroxyphenylethanol + NAD+ + H2O
show the reaction diagram
p-cresol + NADH + H+ + O2
4-methylcatechol + NAD+ + H2O
show the reaction diagram
p-tyrosol + 2 NADH + 2 H+ + O2
3,4-dihydroxyphenylethanol + 2 NAD+ + 2 H2O
show the reaction diagram
phenylethanol + NADH + H+ + O2
m-tyrosol + p-tyrosol + NAD+ + H2O
show the reaction diagram
styrene + NADH + H+ + O2
styrene epoxide + NAD+ + H2O
show the reaction diagram
toluene + NADH + H+ + O2
4-methylphenol + NAD+ + H2O
show the reaction diagram
toluene + NADH + H+ + O2
p-cresol + NAD+ + H2O
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Ferredoxin
-
iron-sulfur centre
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
phenylacetylene
-
selectively inhibits growth on toluene, but not on p-cresol, i.e. the toluene 4-monooxygenase pathway. Inhibition is irreversible
-
TmoD
effector protein
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.011 - 0.068
methyl 4-tolyl sulfide
0.115 - 0.129
methyl phenyl sulfide
0.0023 - 0.009
Toluene
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.16 - 0.23
anisole
0.1 - 0.31
Benzene
0.075 - 0.087
chlorobenzene
0.013 - 0.034
nitrobenzene
0.35 - 0.49
norcarane
0.21 - 3.4
Toluene
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
70 - 1470
Toluene
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.098 - 0.116
TmoD
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
1250
-
pH 7.5, 25C
23890
-
pH 7.5, 25C
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
220000
-
diiron hydrolase T4moH, gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
comparison of X-ray crystal structures of resting and reduced mutant T201A in complex with effector T4moD with structures of wild-type reveals changes in the positions of several key active site residues
-
crystal structures for native T4moD and variants with either four (DELTAN4-), or 10 (DELTAN10-) residues removed from the N-terminal at 2.1-, 1.7-, and 1.9 A resolution, respectively. Alterations of the N-terminal have little influence on the folded core of the protein
fluorescence anisotropy study of the protein-protein interactions. Binding interactions are detected between T4moD and the hydroxylase component T4moH and between T4moD and the Rieske [2Fe-2S] ferredoxin component T4moC. No binding interactions are detected between T4moD and the NADH oxidoreductase component T4moF, but T4moF is able to disrupt binding between T4moC and T4moD
-
high-resolution structures of toluene 4-monooxygenase hydroxylase complexed with its electron transfer protein ferredoxin and comparison with the hydroxylase-effector structure. Ferredoxin or effector protein binding produce different arrangements of conserved residues
-
homology modeling. Residue I100 aids in steering the substrate into the active site at the end of the long entrance channel
solution structure of the T4moD effector protein. The secondary structure of T4moD consists of three alpha-helices and seven beta-strands arranged in an N-terminal betaalphabetabeta and a C-terminal betaalphaalphabetabetabeta domain topology. The region around Asn34 may be involved in structural aspects contributing to functional specificity
structure of toluene 4-monooxygenase ferredoxin oxidoreductase subunit Tmo4F, to 1.6 A resolution. Structure includes ferredoxin, flavin, and NADH binding domains. Close contacts between the C8 methyl group of FAD and [2Fe-2S] ligand Cys36-O represent a pathway for electron transfer between the redox cofactors. Subunit T4moF [2Fe-2S] ligand Cys41 and T4moC [2Fe-2S] ligand His67, along with other electrostatic interactions between the protein partners, form the functional electron transfer interface
structures of toluene 4-monooxygenase hydroxylase in complex with reaction products and effector protein. Active site residue F176 traps the aromatic ring of products against a surface of the active site cavity formed by G103, E104 and A107, while F196 positions the aromatic ring against this surface via a pi-stacking interaction. Effector protein binding produces significant shifts in the positions of residues along the outer portion of the active site (T201, N202, and Q228) and in some iron ligands (E231 and E197), but minor shifts ae produced in F176, F196, and other interior residues of the active site
Q00456 and Q00457 and Q00460 and Q00459
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
partial purification from toluene-grown cells
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expresion in Escherichia coi or Pseudomonas putida
expression in Cupriavidus metallidurans
-
expression in Escherichia coli
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
multicomponent toluene-4-monooxygenase is encoded by a five-gene cluster, tmoABCDE. Each of the five genes is essential for T4MO activity
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
V106A
-
initial thioanisole sulfoxidation is improved by 1.65fold
V106E
-
initial thioanisole sulfoxidation is improved by 1.72fold
V106L
-
initial thioanisole sulfoxidation is decreased by 0.43fold
V106M
-
mutant oxidizes methyl phenyl sulfide to the corresponding sulfoxide at a rate of 3.0 nmol/min/mg protein compared with 1.6 for the wild-type enzyme, and the enantiomeric excess (pro-S) increases from 51% for the wild type to 88% for this mutant. Function of residue V106 is the proper positioning or docking of the substrate with respect to the diiron atoms
V106S
-
initial thioanisole sulfoxidation is decreased by 0.8fold
V106A
-
initial thioanisole sulfoxidation is improved by 1.65fold
-
V106E
-
initial thioanisole sulfoxidation is improved by 1.72fold
-
V106L
-
initial thioanisole sulfoxidation is decreased by 0.43fold
-
V106M
-
mutant oxidizes methyl phenyl sulfide to the corresponding sulfoxide at a rate of 3.0 nmol/min/mg protein compared with 1.6 for the wild-type enzyme, and the enantiomeric excess (pro-S) increases from 51% for the wild type to 88% for this mutant. Function of residue V106 is the proper positioning or docking of the substrate with respect to the diiron atoms
-
V106S
-
initial thioanisole sulfoxidation is decreased by 0.8fold
-
D285A
-
mutation in subunit TmoA, 2.7fold increase in activity with 2-phenylethanol
D285C
-
mutation in subunit TmoA, 4fold increase in activity with 2-phenylethanol
D285I
-
mutation in subunit TmoA, 6.6fold increase in activity with 2-phenylethanol
D285L
-
mutation in subunit TmoA, 5.4fold increase in activity with 2-phenylethanol
D285P
-
mutation in subunit TmoA, 3.3fold increase in activity with 2-phenylethanol
D285Q
-
mutation in subunit TmoA, 10.5fold increase in activity with 2-phenylethanol
D285S
-
mutation in subunit TmoA, 70% of wild-type activity
D285Y mutation in subunit TmoA,
-
3fold increase in activity with 2-phenylethanol
F205I
decrease in regiospecificity for p-cresol formation, about 5-fold increase in the percentage of m-cresol formation. Mutant gives nearly equivalent amounts of benzylic and phenolic products from p-xylene oxidation
G103A/A107S
-
mutation in subunit TmoA, produces 3-methylcatechol (98%) from o-cresol twofold faster and produces 3-methoxycatechol (82%) from 1mM o-methoxyphenol seven times faster than the wild-type
G103S
-
mutation in subunit TmoA, produces 40fold more methoxyhydroquinone from o-methoxyphenol than the wild-type
G103S/A107T
-
mutation in subunit TmoA, produces methylhydroquinone (92%) from o-cresol fourfold faster than wild-type
I100A/D285I
-
mutation in subunit TmoA, 52fold increase in activity with 2-phenylethanol
I100A/D285Q
-
mutation in subunit TmoA, 85fold increase in activity with 2-phenylethanol
I100G/D285I
-
mutation in subunit TmoA, 14.1fold increase in activity with methyl p-tolyl sulfide
I100L/D285S
-
mutation in subunit TmoA, 1.4fold increase in activity with styrene
Q141C
decrease in regiospecificity for p-cresol formation, mutant functions predominantly as an aromatic ring hydroxylase during the oxidation of p-xylene
S395C
Q00456 and Q00457 and Q00460
mutation in subunit TmoA, shows a 15fold increase in 2-phenylethanol hydroxylation rate
T201F
-
mutation causes a substantial shift in the product distribution, and gives o- and p-cresol in a 1:1 ratio
T201K
-
decrease in activity
T201L
Q00456 and Q00457 and Q00460
parameters similar to wild-type
D285A
-
mutation in subunit TmoA, 2.7fold increase in activity with 2-phenylethanol
-
D285P
-
mutation in subunit TmoA, 3.3fold increase in activity with 2-phenylethanol
-
F205I
-
decrease in regiospecificity for p-cresol formation, about 5-fold increase in the percentage of m-cresol formation. Mutant gives nearly equivalent amounts of benzylic and phenolic products from p-xylene oxidation
-
G103A/A107S
-
mutation in subunit TmoA, produces 3-methylcatechol (98%) from o-cresol twofold faster and produces 3-methoxycatechol (82%) from 1mM o-methoxyphenol seven times faster than the wild-type
-
G103S
-
mutation in subunit TmoA, produces 40fold more methoxyhydroquinone from o-methoxyphenol than the wild-type
-
G103S/A107T
-
mutation in subunit TmoA, produces methylhydroquinone (92%) from o-cresol fourfold faster than wild-type
-
I100A
-
mutant hydroxylates o-tyrosol, m-tyrosol and p-tyrosol to form hydroxytyrosol; mutant shows similar rates as wild-type; mutation in subunit TmoA, 35fold increase in activity with 2-phenylethanol
-
I100D
-
mutation improves both reaction rate and enantioselectivity
-
I100G
-
mutant hydroxylates o-tyrosol, m-tyrosol and p-tyrosol to form hydroxytyrosol; mutation increases the wild-type oxidation rate of methyl phenyl sulfide by 1.7fold, and the enantiomeric excess rises from 86% to 98% pro-S. I100G oxidizes methyl para-tolyl sulfide 11 times faster than the wild type does and changes the selectivity from 41% pro-R to 77% pro-S; mutation in subunit TmoA, 11fold increase in activity with methyl p-tolyl sulfide
-
I100L
-
mutation in subunit TmoA, 0.9fold decrease in activity with styrene; mutation in subunit TmoA, produces 3-methoxycatechol from o-methoxyphenol four times faster than wild-type
-
I100S
-
mutant hydroxylates o-tyrosol, m-tyrosol and p-tyrosol to form hydroxytyrosol; mutation improves both reaction rate and enantioselectivity
-
I100V
-
mutant hydroxylates m-tyrosol to form hydroxytyrosol; mutation improves both reaction rate and enantioselectivity
-
Q141C
-
decrease in regiospecificity for p-cresol formation, mutant functions predominantly as an aromatic ring hydroxylase during the oxidation of p-xylene
-
S395C
-
mutation in subunit TmoA, shows a 15fold increase in 2-phenylethanol hydroxylation rate
-
T201A
-
mutant retains catalytic activity and exhibits 80-90% coupling efficiency compared to 94% for wild-type, with p-cresol representing 90-95% of the total product distribution; mutation has no impact on steady-state catalysis or coupling. Mutant T201A gives stoichometric release of H2O2 during reaction in the absence of substrate and has a faster first-order rate constant for product formation than wild-type; parameters similar to wild-type
-
T201F
-
mutation causes a substantial shift in the product distribution, and gives o- and p-cresol in a 1:1 ratio
-
T201G
-
mutant retains catalytic activity and exhibits 80-90% coupling efficiency compared to 94% for wild-type, with p-cresol representing 90-95% of the total product distribution; mutation has no impact on steady-state catalysis or coupling
-
T201K
-
decrease in activity
-
T201L
-
parameters similar to wild-type
-
T201S
-
mutant retains catalytic activity and exhibits 80-90% coupling efficiency compared to 94% for wild-type, with p-cresol representing 90-95% of the total product distribution; mutation has no impact on steady-state catalysis or coupling; parameters similar to wild-type
-
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
synthesis