Information on EC 1.14.13.44 - 2-hydroxybiphenyl 3-monooxygenase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.14.13.44
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RECOMMENDED NAME
GeneOntology No.
2-hydroxybiphenyl 3-monooxygenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
2-hydroxybiphenyl + NADH + H+ + O2 = 2,3-dihydroxybiphenyl + NAD+ + H2O
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
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-
-
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redox reaction
reduction
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-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
2,2'-dihydroxybiphenyl degradation
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2-hydroxybiphenyl degradation
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SYSTEMATIC NAME
IUBMB Comments
2-hydroxybiphenyl,NADH:oxygen oxidoreductase (3-hydroxylating)
Also converts 2,2'-dihydroxybiphenyl into 2,2',3-trihydroxy-biphenyl.
CAS REGISTRY NUMBER
COMMENTARY hide
118251-39-1
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SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2,2'-dihydroxybiphenyl + NADH + O2
2,2',3-trihydroxybiphenyl + NAD+ + H2O
show the reaction diagram
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-
-
-
?
2,2'-dihydroxybiphenyl + NADPH + O2
2,2',3-trihydroxybiphenyl + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
2,3-dihydroxybiphenyl + NADH + H+ + O2
?
show the reaction diagram
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-
-
?
2,5-dihydroxybiphenyl + NADH + O2
2,3,5-trihydroxybiphenyl + NAD+ + H2O
show the reaction diagram
-
-
-
-
?
2-ethylphenol + NADH + O2
1,2-dihydroxy-3-ethylbenzene + NAD+ + H2O
show the reaction diagram
-
-
-
-
?
2-hydroxybiphenyl + NADH + H+ + O2
2,3-dihydroxybiphenyl + NAD+ + H2O
show the reaction diagram
2-hydroxybiphenyl + NADH + O2
2,3-dihydroxybiphenyl + NAD+ + H2O
show the reaction diagram
2-hydroxybiphenyl + NADPH + O2
2,3-dihydroxybiphenyl + NADP+ + H2O
show the reaction diagram
2-hydroxybiphenyl + O2 + NADH + H+
3-phenylcatechol + NAD+ + H2O
show the reaction diagram
2-methylphenol + NADH + O2
1,2-dihydroxy-3-methylbenzene + NAD+ + H2O
show the reaction diagram
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-
-
-
?
2-propylphenol + NADH + O2
1,2-dihydroxy-3-propylbenzene + NAD+ + H2O
show the reaction diagram
2-sec-butylphenol + NADH + O2
2-sec-butylcatechol + NAD+ + H2O
show the reaction diagram
2-sec-butylphenol + NADPH + O2
2-sec-butylcatechol + NADP+ + H2O
show the reaction diagram
-
-
-
-
?
2-tert-butylphenol + NADH + O2
1,2-dihydroxy-3-tert-butylbenzene + NAD+ + H2O
show the reaction diagram
guaiacol + NADH + O2
2,3-dihydroxy-methoxybenzene + NAD+ + H2O
show the reaction diagram
indole + NADH + O2
?
show the reaction diagram
additional information
?
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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
2-hydroxybiphenyl + NADH + O2
2,3-dihydroxybiphenyl + NAD+ + H2O
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NADPH
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can replace NADH as electron donor, Km-value for NADPH is much higher than for NADH
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2,3-Dihydroxybiphenyl
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inhibits reaction with 2-hydroxybiphenyl
AgNO3
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0.01 mM, complete inhibition
CuSO4
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0.01 mM, complete inhibition
FeSO4
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0.08 mM, 30% inhibition
HgCl2
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0.01 mM, complete inhibition
NaCl
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10 mM, 36% inhibition. 100 mM, 89% inhibition
p-hydroxymercuribenzoate
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partial protection in presence of 2-hydroxybiphenyl, reversed by excess of dithiothreitol
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0034 - 0.004
2,2'-Dihydroxybiphenyl
0.0019 - 0.0295
2-Hydroxybiphenyl
0.0057
2-sec-Butylphenol
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reaction with NADH or NADPH and O2
0.0097 - 0.531
NADH
0.0943 - 0.137
NADPH
0.0292
O2
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reaction with 2-hydroxybiphenyl and NADH
additional information
additional information
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Km-values for wild-type and mutant enzymes
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
9 - 9.4
2,2'-Dihydroxybiphenyl
0.15 - 15.6
2-Hydroxybiphenyl
10.2 - 15.8
2-sec-Butylphenol
0.95
guaiacol
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-
0.005 - 0.09
indole
0.14 - 16.2
NADH
11.2 - 18.8
NADPH
16.2
O2
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reaction with 2-hydroxybiphenyl and NADH
additional information
additional information
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turnover-numbers for mutant enzymes
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5.4 - 730
2-Hydroxybiphenyl
0.314 - 57
NADH
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.9
2,3-Dihydroxybiphenyl
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-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.2 - 7.8
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more than 80% of maximal activity at pH 7.2 and pH 7.8, beyond pH 7.8 activity declines abruptly with increasing pH
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
PDB
SCOP
CATH
UNIPROT
ORGANISM
Pseudomonas nitroreducens;
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60000
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4 * 60000, SDS-PAGE
256000
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
tetramer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging drop method, structure of the enzyme with bound 2-hydroxybiphenyl, as well as several variants, at a resolution of 2.3-2.5 A to investigate structure function correlations of the enzyme. An observed hydrogen bond between 2-hydroxybiphenyl and His48 in the active site confirms the role of this residue in substrate deprotonation. The entrance to the active site is confirmed by generating variant G255F which exhibits only 7% of the wild-type's specific activity of product formation, suggesting inhibition of substrate entrance into the active site by the large aromatic residue. Residue Arg242 is suggested to facilitate FAD movement and reduction as was previously reported in studies on the homologous protein para-hydroxybenzoate hydroxylase. In addition, it is suggested that Trp225,which is located in the active site, facilitates proper substrate entrance into the binding pocket in contrast to aklavinone-11-hydroxylase and para-hydroxybenzoate hydroxylase in which a residue at a similar position is responsible for substrate deprotonation. Structure function correlations described in this work will aid in the design of variants with improved activity and altered selectivity for potential industrial applications
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hanging-drop vapour-diffusion, optimized precipitant solution contains 1.6 M ammonium sulfate, 100 mM sodium chloride and 100 mM MES-NaOH, pH 7.5, crystals of native and SeMet-HbpA diffract to 2.01 and 2.25 A, respectively
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single crystals of the enzyme are used to determine its structure in two forms: an FAD-bound form that allows characterisation of the active site, and an apo form that, although lacking flavin, gives extra information on the location of residues and structure of mobile loops that are absent from the FAD complex
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, pure enzyme at concentration of 3.8 mg/ml in 50 mM phosphate buffer, pH 7.5, stable for at least 6 months
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
native and recombinant enzyme
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recombinant enzyme
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recombinant HbpA
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
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expression in Escherichia coli BL21
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
G255F
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the variant exhibits 7% compared to the specific activity of the wild-type enzyme on NADH and 2,3-dihydroxybiphenyl, suggesting inhibition of substrate entrance into the active site by the large aromatic residue
I244V
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mutant enzyme has a 30% higher specific activity with 2-sec-butylphenol, guaiacol, and 2-hydroxybiphenyl. The Km-value for guaiacol decreases with this mutant, but the Km-value for 2-hydroxybiphenyl increase
R242A
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the variant is not active on NADH and 2,3-dihydroxybiphenyl
R242E
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the variant is not active on NADH and 2,3-dihydroxybiphenyl
R242Q
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the variant is not active on NADH and 2,3-dihydroxybiphenyl
V368A/L417F
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double replacement improves the efficiency of substrate hydroxylation by reducing the uncoupled oxidation of NADH. With guaiacol as substrate, the Vmax is increased and the Km-value is decreased. With 2-tert-butylphenol as substrate the turnover number is increased more than 5fold
W225A
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the variant exhibits 7% compared to the specific activity of the wild-type enzyme on NADH and 2,3-dihydroxybiphenyl
W225Y
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the variant exhibits 122% compared to the specific activity of the wild-type enzyme on NADH and 2,3-dihydroxybiphenyl
I244V
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mutant enzyme has a 30% higher specific activity with 2-sec-butylphenol, guaiacol, and 2-hydroxybiphenyl. The Km-value for guaiacol decreases with this mutant, but the Km-value for 2-hydroxybiphenyl increase
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V368A/L417F
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double replacement improves the efficiency of substrate hydroxylation by reducing the uncoupled oxidation of NADH. With guaiacol as substrate, the Vmax is increased and the Km-value is decreased. With 2-tert-butylphenol as substrate the turnover number is increased more than 5fold
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additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis