Information on EC 1.14.14.9 - 4-hydroxyphenylacetate 3-monooxygenase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.14.14.9
-
RECOMMENDED NAME
GeneOntology No.
4-hydroxyphenylacetate 3-monooxygenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
4-hydroxyphenylacetate + FADH2 + O2 = 3,4-dihydroxyphenylacetate + FAD + H2O
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydroxylation
-
-
-
-
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
4-hydroxyphenylacetate degradation
-
-
aromatic biogenic amine degradation (bacteria)
-
-
Microbial metabolism in diverse environments
-
-
Tyrosine metabolism
-
-
4-hydroxyphenylacetate degradation
-
-
SYSTEMATIC NAME
IUBMB Comments
4-hydroxyphenylacetate,FAD:oxygen oxidoreductase (3-hydroxylating)
The enzyme from Escherichia coli attacks a broad spectrum of phenolic compounds. The enzyme uses FADH2 as a substrate rather than a cofactor [4]. FADH2 is provided by EC 1.5.1.36, flavin reductase (NADH) [5,6].
CAS REGISTRY NUMBER
COMMENTARY hide
37256-71-6
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
wild-type strain 3B-1 and mutant 3B-3
-
-
Manually annotated by BRENDA team
PRL W15
-
-
Manually annotated by BRENDA team
PRL W15
-
-
Manually annotated by BRENDA team
strain C
-
-
Manually annotated by BRENDA team
strain PA-9, gene hpaH
UniProt
Manually annotated by BRENDA team
strain PA-9, gene hpaH
UniProt
Manually annotated by BRENDA team
strain HTB24
-
-
Manually annotated by BRENDA team
strain HTB24
-
-
Manually annotated by BRENDA team
no activity in Escherichia coli K-12
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2,4-dihydroxybenzoate
?
show the reaction diagram
-
-
-
-
?
2,5-dihydroxyphenylacetate + ?
?
show the reaction diagram
-
155% of 4-hydroxyphenylacetate activity
-
-
?
2-phenylbutyric acid + H2
?
show the reaction diagram
-
86% of 4-hydroxyphenylacetate activity
-
-
?
2-phenylpropionic acid + ?
?
show the reaction diagram
-
77% of 4-hydroxyphenylacetate activity
-
-
?
3,4-dihydroxybenzoate + ?
?
show the reaction diagram
-
30% of 4-hydroxyphenylacetate activity
-
-
?
3,4-dihydroxyphenylacetate + ?
?
show the reaction diagram
3-(4-hydroxyphenyl)propionate + ?
3-(3,4-dihydroxy)phenylpropionate + ?
show the reaction diagram
3-hydroxyphenylacetate + FMNH2 + O2
3,4-hydroxyphenylacetate + FMN + H2O
show the reaction diagram
3-hydroxyphenylacetate + NAD(P)H + O2
3,4-hydroxyphenylacetate + NAD(P)+ + H2O
show the reaction diagram
3-hydroxyphenylacetate + NADH + O2
3,4-hydroxyphenylacetate + NAD+ + H2O
show the reaction diagram
4-aminobenzoate + ?
4-amino-3-hydroxybenzoate + ?
show the reaction diagram
-
-
-
?
4-aminophenylacetate + ?
4-amino-3-hydroxyphenylacetate + ?
show the reaction diagram
4-bromophenol + NADH + H+ + O2
4-bromocatechol + NAD+ + H2O
show the reaction diagram
rate of halophenol consumption 2.9 microM/min/g cell dry weight
-
-
?
4-chlorophenol + NADH + H+ + O2
4-chlorocatechol + NAD+ + H2O
show the reaction diagram
rate of halophenol consumption 3.3 microM/min/g cell dry weight
-
-
?
4-coumaric acid + FADH2 + O2
caffeic acid + FAD + H2O
show the reaction diagram
4-fluorophenol + NADH + H+ + O2
4-fluorocatechol + NAD+ + H2O
show the reaction diagram
rate of halophenol consumption 5.3 microM/min/g cell dry weight
-
-
?
4-fluorophenylacetate + NADH + O2
4-fluoro-3-hydroxyphenylacetate + NAD+ + H2O
show the reaction diagram
-
-
-
?
4-hydroxybenzoate + ?
3,4-dihydroxybenzoate + ?
show the reaction diagram
-
15% of 4-hydroxyphenylacetate activity
-
?
4-hydroxyphenylacetate + FADH2 + O2
3,4-dihydroxyphenylacetate + FAD + H2O
show the reaction diagram
4-hydroxyphenylacetate + FMNH + O2
3,4-dihydroxyphenylacetate + FMN + H2O
show the reaction diagram
-
-
-
-
?
4-hydroxyphenylacetate + FMNH2 + O2
3,4-dihydroxyphenylacetate + FMN + H2O
show the reaction diagram
-
-
-
-
?
4-hydroxyphenylacetate + NAD(P)H + H+ + O2
3,4-dihydroxyphenylacetate + NAD(P)+ + H2O
show the reaction diagram
4-hydroxyphenylacetate + NAD(P)H + O2
3,4-dihydroxyphenylacetate + NAD(P)+ + H2O
show the reaction diagram
4-hydroxyphenylacetate + NADH + H+ + O2
3,4-dihydroxyphenylacetate + NAD+ + H2O
show the reaction diagram
4-hydroxyphenylacetate + NADH + O2
3,4-dihydroxyphenylacetate + NAD+ + H2O
show the reaction diagram
4-hydroxyphenylacetate + NADPH + H+ + O2
3,4-dihydroxyphenylacetate + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
4-hydroxyphenylacetic acid + O2 + NADH + H+
3,4-dihydroxyphenylacetic acid + H2O + NAD+
show the reaction diagram
4-hydroxyphenylethanol + O2 + NADH + H+
3,4-dihydroxyphenylethanol + H2O + NAD+
show the reaction diagram
4-iodophenol + NADH + H+ + O2
4-iodocatechol + NAD+ + H2O
show the reaction diagram
rate of halophenol consumption 1.7 microM/min/g cell dry weight
-
-
?
catechol + ?
?
show the reaction diagram
-
30% of 4-hydroxyphenylacetate activity
-
-
?
L-(3,4-dihydroxy)phenylalanine + ?
?
show the reaction diagram
-
10% of 4-hydroxyphenylacetate
-
?
L-tyrosine + ?
L-(3,4-dihydroxy)phenylalanine + ?
show the reaction diagram
-
8% of 4-hydroxyphenylacetate activity
-
?
L-tyrosine + FADH2 + O2
L-(3,4-dihydroxy)phenylalanine + FAD + H2O
show the reaction diagram
p-cresol + ?
?
show the reaction diagram
-
51% of 4-hydroxyphenylacetate activity
-
-
?
phenol + ?
catechol + ?
show the reaction diagram
phenylacetic acid + ?
?
show the reaction diagram
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
4-hydroxyphenylacetate + FADH2 + O2
3,4-dihydroxyphenylacetate + FAD + H2O
show the reaction diagram
4-hydroxyphenylacetate + FMNH + O2
3,4-dihydroxyphenylacetate + FMN + H2O
show the reaction diagram
-
-
-
-
?
4-hydroxyphenylacetate + FMNH2 + O2
3,4-dihydroxyphenylacetate + FMN + H2O
show the reaction diagram
-
-
-
-
?
4-hydroxyphenylacetate + NAD(P)H + H+ + O2
3,4-dihydroxyphenylacetate + NAD(P)+ + H2O
show the reaction diagram
4-hydroxyphenylacetate + NADH + H+ + O2
3,4-dihydroxyphenylacetate + NAD+ + H2O
show the reaction diagram
4-hydroxyphenylacetate + NADH + O2
3,4-dihydroxyphenylacetate + NAD+ + H2O
show the reaction diagram
4-hydroxyphenylacetate + NADPH + H+ + O2
3,4-dihydroxyphenylacetate + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
FADH2
flavin
-
dependent on
FMNH2
NAD(P)H
NADPH
riboflavin
additional information
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Br-
-
retards the oxidative half-reaction
Cl-
-
retards the oxidative half-reaction
F-
-
retards the oxidative half-reaction
I-
-
retards the oxidative half-reaction
N3-
-
retards the oxidative half-reaction
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4-hydroxyphenylacetate
Cibacron blue F3GA
EDTA
-
inhibits non-purified enzyme, ammonium sulfate precipitate
FAD
-
inhibitory effect at more than 0.15 mM
FMN
-
inhibitory effect at more than 0.15 mM
iodoacetate
-
-
N-ethylmaleimide
o-phenanthroline
-
complete inhibition at 1 mM
p-chloromercuribenzoate
-
92% inhibition at 1 mM
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4-hydroxyphenylacetate
-
-
Carbohydrates
-
stimulate synthesis of enzyme insignificantly
-
additional information
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.014 - 26
4-hydroxyphenylacetate
0.0031 - 0.0042
FAD
0.0021
FMN
-
-
0.012 - 28
NADH
0.16
phenylacetic acid
-
-
0.0026
riboflavin
-
-
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.833 - 389
4-hydroxyphenylacetate
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.026
-
with 3-hydroxyphenylacetate as substrate
0.028
-
with 4-hydroxyphenylacetate as substrate
0.14
-
activity of HpaB protein in presence of NADH and HpaC protein
0.191
-
strain W, induced by 4-hydroxyphenylacetate
0.231
-
for 4-hydroxyphenylacetate oxidation
3.4
-
purified enzyme
8.3
purified recombinant C2 component; purified recombinant C2 component
8.89
-
C2 component
200
purified recombinant C1 component; purified recombinant C1 component
201
-
C1 component
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.2
-
assay at
7.8
-
assay at
8
-
assay at
additional information
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 8
-
more than 80% of activity
6.2 - 9.9
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assay range for determination of reaction kinetics of C2-FMNH with oxygen at various pH values investigated by stopped-flow and rapid quenched-flow techniques, detailed overview
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
24
-
assay at
28
-
assay at
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Brucella melitensis biotype 1 (strain 16M / ATCC 23456 / NCTC 10094)
Brucella melitensis biotype 1 (strain 16M / ATCC 23456 / NCTC 10094)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
18679
-
x * 18679, SDS-PAGE, enzyme is needed for activity
18680
-
x * 58781 flavoprotein HpaA, x * 18680, coupling protein, estimation from gene sequence
19000
-
2 * 19000 small component, 2 * 59000 large component
30750
-
2 * 30750, small component
32000
-
x * 32000, small component, SDS-PAGE
32500
-
2 * 32500, alpha2 oligomeric structure, SDS-PAGE
35000
x * 35000, C1 component, SDS-PAGE, x * 47000, C2 component, SDS-PAGE; x * 35000, C1 component, SDS-PAGE, x * 47000, C2 component, SDS-PAGE
38500
-
1 * 38500, large component
47000
x * 35000, C1 component, SDS-PAGE, x * 47000, C2 component, SDS-PAGE; x * 35000, C1 component, SDS-PAGE, x * 47000, C2 component, SDS-PAGE
50000
-
homotetramer, 4 * 50000, large component, SDS-PAGE
56000
x * 56269, sequence calculation, x * 56000, recombinant enzyme, SDS-PAGE
56269
x * 56269, sequence calculation, x * 56000, recombinant enzyme, SDS-PAGE
58500
-
x * 58500, SDS-PAGE
65000
-
gel filtration
73000
-
component C1, gel filtration
91000
-
gel filtration
120000
-
gel filtration
209000
-
component C2, gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
-
1 * 38500, large component
tetramer
-
homotetramer, 4 * 50000, large component, SDS-PAGE
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
microbatch method, using 20% (W/v) PEG 400, 0.1 M sodium acetate pH 4.6 as a precipitant
-
crystal structures of ligand-free form, a binary complex with FAD, and a ternary complex with FAD and 4-hydroxyphenylacetate, to 2.0, 1.66 and 1.66 A resolution, respectively. Binding and dissociation of flavin are accompanied by conformational changes of the loop between beta5 and beta6 and of the loop between beta8 and beta9, leading to preformation of part of the substrate-binding site Ser197 and Thr198. The latter loop further changes its conformation upon binding of 4-hydroxyphenylacetate and obstructs the active site from the bulk solvent. Arg100 is located adjacent to the putative oxygen-binding site; HpaB in three states: a ligand-free form, a binary complex with FAD, and a ternary complex with FAD and 4-hydroxyphenylacetate, mixing of 0.004 ml of protein solution containing 5 mg/ml protein and 1 mM DTT, with an equal volume of reservoir solution containing 1.5 M ammonium sulfate, 0.1 M Tris-HCl, pH 8.5, and 25% v/v glycerol, crystals appear within a few min and grow during 1-4 days to final size, complex crystal formation by soaking of crystals in 5 mM ligand containing solutions, X-ray diffraction structure determination and analysis at 1.66-2.07 A resolution
-
flavin reductase component HpaC of 4-hydroxyphenylacetate 3-monooxygenase in a ternary complex with FAD and NAD+, mixing of 0.002-0.005 ml of a solution containing 25 mg/mL HpaCTt-FAD, 5 mM NADH, and 1 mM dithiothreitol with an equal volume of a reservoir solution containing 20% w/v PEG 1000, 10% w/v PEG 8000, and 10% v/v glycerol, and then equilibrating the solution against the reservoir solution, 2 days, X-ray diffraction structure determination and analysis at 1.65-3.3 A resolution
-
sitting drop vapour diffusion method, 10 mg/ml protein in 0.01 M Tris-HCl, pH 7.6, equilibration against 0.5 ml of reservoir solution, different mixtures, overview, 20°C, well shaped single crystals, X-ray diffraction structure determination and analysis at 1.82 A resolution
-
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4
-
15-20 days
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
HpaC consists of low stability
-
rather unstable in solution
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, HpaC, small protein, very stable, no significant loss of activity during 2 months
-
-20°C, stable for at least 5 months
-
4°C, 15-20 days
-
4°C, 20 mM phosphate buffer, pH 8.0, concentrated with polyethylene glycol 20000
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
affinity chromatography, gel filtration
-
ammonium sulfate precipitation, DEAE-Sepharose column chromatography, phenyl Sepharose column chromatography, and G-25 gel filtration
-
ammonium sulfate precipitation, gel filtration, affinity chromatography
-
ammonium sulfate precipitation, ion exchange
-
ammonium sulfate precipitation, ion-exchange, gel filtration
-
ammonium sulfate precipitation, ion-exchange, gel filtration. Affinity chromatography for the expressed cholin-binding domain containing HpaB protein
-
difficult because of the low stability of the enzyme in solution
-
from membranes by solubilization with 0.1% Triton X-100, anion exchange chromatography, dialysis, exchange of detergent to dodecyl-beta-D-maltoside, another ion exchange chromatography, and gel filtration, further purification of HpaB by repetitive crystallization, method overview
-
protamine sulfate and ammonium sulfate
-
protamine sulfate precipitation, ion-exchange, gel filtration
-
recombinant enzyme components C1 and C2 from Escherichia coli strain BL21(DE3), C1 4.0fold, C2 1.81fold; recombinant enzyme components C1 and C2 from Escherichia coli strain BL21(DE3), C1 4.0fold, C2 1.81fold
recombinant enzyme from Escherichia coli strain DH5alpha by affinity chromatography on a 4-hydroxyphenylacetate-coupled amino-agarose column to near homogeneity
ultracentrifugation, ammonium sulfate precipitation
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21 Star (DE3) cells
-
expressed in Escherichia coli BL21(DE3) cells
-
expressed in Escherichia coli DH1
-
expressed in Escherichia coli DH1 and TG1
-
expression in Escherichia coli
expression in Escherichia coli K-12
-
gene hpaB (oxygenase component), gene hbaC (reductase component), expressed in Escherichia coli
-
gene hpaH, DNA and amino acid sequence determination and analysis, sequence comparisons, expression in enzyme-deficient Escherichia coli strain DH5alpha
genes c1-hpah and c2-hpah, DNA and amino acid sequence determination and analysis, expression in Escherichia coli strain BL21(DE3), the recombinant enzyme is similarly active cmpared to the native one; genes c1-hpah and c2-hpah, DNA and amino acid sequence determination and analysis, expression in Escherichia coli strain BL21(DE3), the recombinant enzyme is similarly active compared to the native one
HpaA and HpaH are expressed in Escherichia coli W-21, CC118 and YS1 as well as Klebsiella pneumonia mutant strain AG813
-
hpaB gene is expressed in Escherichia coli BL21(DE3)
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
H120D
-
mutant can form C4a-hydroperoxy-FMN, a reactive intermediate necessary for hydroxylation, but cannot hydroxylate 4-hydroxyphenylacetate
H120E
-
mutant can form C4a-hydroperoxy-FMN, a reactive intermediate necessary for hydroxylation, but cannot hydroxylate 4-hydroxyphenylacetate
H120K
-
catalyzes hydroxylation with efficiency comparable to that of the wild-type enzyme, the hydroxylation rate constant for H120K is 5.7 per s and the product conversion ratio is 75%, compared to values of 16 s-1 and 90% for the wild-type enzyme
H120N
-
mutant can form C4a-hydroperoxy-FMN, a reactive intermediate necessary for hydroxylation, but cannot hydroxylate 4-hydroxyphenylacetate
H120Q
-
mutant can form C4a-hydroperoxy-FMN, a reactive intermediate necessary for hydroxylation, but cannot hydroxylate 4-hydroxyphenylacetate
H120R
-
mutant is able to catalyze hydroxylation
H120Y
-
mutant can form C4a-hydroperoxy-FMN, a reactive intermediate necessary for hydroxylation, but cannot hydroxylate 4-hydroxyphenylacetate
S146A
-
product formation is pH-independent and constant at about 70% over a pH range of 6-10
S146C
-
product formation decreases from about 65% at pH 6.0 to 27% at pH 10.0
S171A
-
the mutant shows reduced activity compared to the wild type enzyme
S171A/H396V
-
inactive
S171T
-
the mutant shows about wild type activity
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis
enzyme completely transforms 4-substituted halophenols to 4-halocatechols at 2 mM within a 1-2 h period. An increase in 4-halophenol concentration to 4.8 mM results in a 2.5-20fold decrease in biotransformation efficiency depending on the substrate tested. Organic solvent extraction of the 4-halocatechol products followed by column chromatography results in the production of purified products with a final yield of between 33% and 38%
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