BRENDA - Enzyme Database
show all sequences of 1.14.13.44

A crystal structure of 2-hydroxybiphenyl 3-monooxygenase with bound substrate provides insights into the enzymatic mechanism

Kanteev, M.; Bregman-Cohen, A.; Deri, B.; Shahar, A.; Adir, N.; Fishman, A.; Biochim. Biophys. Acta 1854, 1906-1913 (2015)

Data extracted from this reference:

Cloned(Commentary)
Cloned (Commentary)
Organism
expression in Escherichia coli
Pseudomonas nitroreducens
Crystallization (Commentary)
Crystallization (Commentary)
Organism
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
Pseudomonas nitroreducens
Engineering
Protein Variants
Commentary
Organism
G255F
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
Pseudomonas nitroreducens
R242A
the variant is not active on NADH and 2,3-dihydroxybiphenyl
Pseudomonas nitroreducens
R242E
the variant is not active on NADH and 2,3-dihydroxybiphenyl
Pseudomonas nitroreducens
R242Q
the variant is not active on NADH and 2,3-dihydroxybiphenyl
Pseudomonas nitroreducens
W225A
the variant exhibits 7% compared to the specific activity of the wild-type enzyme on NADH and 2,3-dihydroxybiphenyl
Pseudomonas nitroreducens
W225Y
the variant exhibits 122% compared to the specific activity of the wild-type enzyme on NADH and 2,3-dihydroxybiphenyl
Pseudomonas nitroreducens
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.0031
-
2-Hydroxybiphenyl
pH 7.5, 30°C, wild-type enzyme
Pseudomonas nitroreducens
0.0228
-
2-Hydroxybiphenyl
pH 7.5, 30°C, mutant enzyme W225Y
Pseudomonas nitroreducens
0.0266
-
2-Hydroxybiphenyl
pH 7.5, 30°C, mutant enzyme G255F
Pseudomonas nitroreducens
0.0278
-
2-Hydroxybiphenyl
pH 7.5, 30°C, mutant enzyme R242E
Pseudomonas nitroreducens
0.0295
-
2-Hydroxybiphenyl
pH 7.5, 30°C, mutant enzyme W225A
Pseudomonas nitroreducens
0.102
-
NADH
pH 7.5, 30°C, mutant enzyme W225Y
Pseudomonas nitroreducens
0.149
-
NADH
pH 7.5, 30°C, wild-type enzyme
Pseudomonas nitroreducens
0.222
-
NADH
pH 7.5, 30°C, mutant enzyme R242Q
Pseudomonas nitroreducens
0.253
-
NADH
pH 7.5, 30°C, mutant enzyme W225A
Pseudomonas nitroreducens
0.336
-
NADH
pH 7.5, 30°C, mutant enzyme R242A
Pseudomonas nitroreducens
0.427
-
NADH
pH 7.5, 30°C, mutant enzyme R242E
Pseudomonas nitroreducens
0.531
-
NADH
pH 7.5, 30°C, mutant enzyme G255F
Pseudomonas nitroreducens
Organism
Organism
UniProt
Commentary
Textmining
Pseudomonas nitroreducens
O06647
-
-
Purification (Commentary)
Purification (Commentary)
Organism
-
Pseudomonas nitroreducens
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
Substrate Product ID
2,3-dihydroxybiphenyl + NADH + H+ + O2
-
744407
Pseudomonas nitroreducens
?
-
-
-
?
2-hydroxybiphenyl + NADH + H+ + O2
it is suggested that Trp225, which is located in the active site, facilitates proper substrate entrance into the binding pocket
744407
Pseudomonas nitroreducens
2,3-dihydroxybiphenyl + NAD+ + H2O
-
-
-
?
Synonyms
Synonyms
Commentary
Organism
2-hydroxybiphenyl 3-monooxygenase
-
Pseudomonas nitroreducens
HbpA
-
Pseudomonas nitroreducens
Turnover Number [1/s]
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
0.14
-
NADH
pH 7.5, 30°C, mutant enzyme R242A
Pseudomonas nitroreducens
0.15
-
NADH
pH 7.5, 30°C, mutant enzyme R242E
Pseudomonas nitroreducens
0.15
-
2-Hydroxybiphenyl
pH 7.5, 30°C, mutant enzyme R242E
Pseudomonas nitroreducens
0.24
-
NADH
pH 7.5, 30°C, mutant enzyme R242Q
Pseudomonas nitroreducens
0.46
-
2-Hydroxybiphenyl
pH 7.5, 30°C, mutant enzyme W225A
Pseudomonas nitroreducens
0.71
-
2-Hydroxybiphenyl
pH 7.5, 30°C, mutant enzyme G255F
Pseudomonas nitroreducens
1.34
-
NADH
pH 7.5, 30°C, mutant enzyme G255F
Pseudomonas nitroreducens
2.26
-
2-Hydroxybiphenyl
pH 7.5, 30°C, wild-type enzyme
Pseudomonas nitroreducens
3.16
-
NADH
pH 7.5, 30°C, wild-type enzyme
Pseudomonas nitroreducens
3.45
-
NADH
pH 7.5, 30°C, mutant enzyme W225A
Pseudomonas nitroreducens
4.51
-
2-Hydroxybiphenyl
pH 7.5, 30°C, mutant enzyme W225Y
Pseudomonas nitroreducens
5.8
-
NADH
pH 7.5, 30°C, mutant enzyme W225Y
Pseudomonas nitroreducens
Cofactor
Cofactor
Commentary
Organism
Structure
FAD
residue Arg242 is suggested to facilitate FAD movement and reduction
Pseudomonas nitroreducens
Cloned(Commentary) (protein specific)
Commentary
Organism
expression in Escherichia coli
Pseudomonas nitroreducens
Cofactor (protein specific)
Cofactor
Commentary
Organism
Structure
FAD
residue Arg242 is suggested to facilitate FAD movement and reduction
Pseudomonas nitroreducens
Crystallization (Commentary) (protein specific)
Crystallization
Organism
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
Pseudomonas nitroreducens
Engineering (protein specific)
Protein Variants
Commentary
Organism
G255F
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
Pseudomonas nitroreducens
R242A
the variant is not active on NADH and 2,3-dihydroxybiphenyl
Pseudomonas nitroreducens
R242E
the variant is not active on NADH and 2,3-dihydroxybiphenyl
Pseudomonas nitroreducens
R242Q
the variant is not active on NADH and 2,3-dihydroxybiphenyl
Pseudomonas nitroreducens
W225A
the variant exhibits 7% compared to the specific activity of the wild-type enzyme on NADH and 2,3-dihydroxybiphenyl
Pseudomonas nitroreducens
W225Y
the variant exhibits 122% compared to the specific activity of the wild-type enzyme on NADH and 2,3-dihydroxybiphenyl
Pseudomonas nitroreducens
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.0031
-
2-Hydroxybiphenyl
pH 7.5, 30°C, wild-type enzyme
Pseudomonas nitroreducens
0.0228
-
2-Hydroxybiphenyl
pH 7.5, 30°C, mutant enzyme W225Y
Pseudomonas nitroreducens
0.0266
-
2-Hydroxybiphenyl
pH 7.5, 30°C, mutant enzyme G255F
Pseudomonas nitroreducens
0.0278
-
2-Hydroxybiphenyl
pH 7.5, 30°C, mutant enzyme R242E
Pseudomonas nitroreducens
0.0295
-
2-Hydroxybiphenyl
pH 7.5, 30°C, mutant enzyme W225A
Pseudomonas nitroreducens
0.102
-
NADH
pH 7.5, 30°C, mutant enzyme W225Y
Pseudomonas nitroreducens
0.149
-
NADH
pH 7.5, 30°C, wild-type enzyme
Pseudomonas nitroreducens
0.222
-
NADH
pH 7.5, 30°C, mutant enzyme R242Q
Pseudomonas nitroreducens
0.253
-
NADH
pH 7.5, 30°C, mutant enzyme W225A
Pseudomonas nitroreducens
0.336
-
NADH
pH 7.5, 30°C, mutant enzyme R242A
Pseudomonas nitroreducens
0.427
-
NADH
pH 7.5, 30°C, mutant enzyme R242E
Pseudomonas nitroreducens
0.531
-
NADH
pH 7.5, 30°C, mutant enzyme G255F
Pseudomonas nitroreducens
Purification (Commentary) (protein specific)
Commentary
Organism
-
Pseudomonas nitroreducens
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
ID
2,3-dihydroxybiphenyl + NADH + H+ + O2
-
744407
Pseudomonas nitroreducens
?
-
-
-
?
2-hydroxybiphenyl + NADH + H+ + O2
it is suggested that Trp225, which is located in the active site, facilitates proper substrate entrance into the binding pocket
744407
Pseudomonas nitroreducens
2,3-dihydroxybiphenyl + NAD+ + H2O
-
-
-
?
Turnover Number [1/s] (protein specific)
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
0.14
-
NADH
pH 7.5, 30°C, mutant enzyme R242A
Pseudomonas nitroreducens
0.15
-
NADH
pH 7.5, 30°C, mutant enzyme R242E
Pseudomonas nitroreducens
0.15
-
2-Hydroxybiphenyl
pH 7.5, 30°C, mutant enzyme R242E
Pseudomonas nitroreducens
0.24
-
NADH
pH 7.5, 30°C, mutant enzyme R242Q
Pseudomonas nitroreducens
0.46
-
2-Hydroxybiphenyl
pH 7.5, 30°C, mutant enzyme W225A
Pseudomonas nitroreducens
0.71
-
2-Hydroxybiphenyl
pH 7.5, 30°C, mutant enzyme G255F
Pseudomonas nitroreducens
1.34
-
NADH
pH 7.5, 30°C, mutant enzyme G255F
Pseudomonas nitroreducens
2.26
-
2-Hydroxybiphenyl
pH 7.5, 30°C, wild-type enzyme
Pseudomonas nitroreducens
3.16
-
NADH
pH 7.5, 30°C, wild-type enzyme
Pseudomonas nitroreducens
3.45
-
NADH
pH 7.5, 30°C, mutant enzyme W225A
Pseudomonas nitroreducens
4.51
-
2-Hydroxybiphenyl
pH 7.5, 30°C, mutant enzyme W225Y
Pseudomonas nitroreducens
5.8
-
NADH
pH 7.5, 30°C, mutant enzyme W225Y
Pseudomonas nitroreducens
KCat/KM [mM/s]
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
0.314
-
NADH
pH 7.5, 30°C, mutant enzyme R242A
Pseudomonas nitroreducens
0.351
-
NADH
pH 7.5, 30°C, mutant enzyme R242E
Pseudomonas nitroreducens
1.08
-
NADH
pH 7.5, 30°C, mutant enzyme R242Q
Pseudomonas nitroreducens
2.53
-
NADH
pH 7.5, 30°C, mutant enzyme G255F
Pseudomonas nitroreducens
5.4
-
2-Hydroxybiphenyl
pH 7.5, 30°C, mutant enzyme R242A
Pseudomonas nitroreducens
14
-
NADH
pH 7.5, 30°C, mutant enzyme W225A
Pseudomonas nitroreducens
20
-
NADH
pH 7.5, 30°C, wild-type enzyme
Pseudomonas nitroreducens
20
-
2-Hydroxybiphenyl
pH 7.5, 30°C, mutant enzyme W225A
Pseudomonas nitroreducens
30
-
2-Hydroxybiphenyl
pH 7.5, 30°C, mutant enzyme G255F
Pseudomonas nitroreducens
57
-
NADH
pH 7.5, 30°C, mutant enzyme W225Y
Pseudomonas nitroreducens
200
-
2-Hydroxybiphenyl
pH 7.5, 30°C, mutant enzyme W225Y
Pseudomonas nitroreducens
730
-
2-Hydroxybiphenyl
pH 7.5, 30°C, wild-type enzyme
Pseudomonas nitroreducens
KCat/KM [mM/s] (protein specific)
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
0.314
-
NADH
pH 7.5, 30°C, mutant enzyme R242A
Pseudomonas nitroreducens
0.351
-
NADH
pH 7.5, 30°C, mutant enzyme R242E
Pseudomonas nitroreducens
1.08
-
NADH
pH 7.5, 30°C, mutant enzyme R242Q
Pseudomonas nitroreducens
2.53
-
NADH
pH 7.5, 30°C, mutant enzyme G255F
Pseudomonas nitroreducens
5.4
-
2-Hydroxybiphenyl
pH 7.5, 30°C, mutant enzyme R242A
Pseudomonas nitroreducens
14
-
NADH
pH 7.5, 30°C, mutant enzyme W225A
Pseudomonas nitroreducens
20
-
NADH
pH 7.5, 30°C, wild-type enzyme
Pseudomonas nitroreducens
20
-
2-Hydroxybiphenyl
pH 7.5, 30°C, mutant enzyme W225A
Pseudomonas nitroreducens
30
-
2-Hydroxybiphenyl
pH 7.5, 30°C, mutant enzyme G255F
Pseudomonas nitroreducens
57
-
NADH
pH 7.5, 30°C, mutant enzyme W225Y
Pseudomonas nitroreducens
200
-
2-Hydroxybiphenyl
pH 7.5, 30°C, mutant enzyme W225Y
Pseudomonas nitroreducens
730
-
2-Hydroxybiphenyl
pH 7.5, 30°C, wild-type enzyme
Pseudomonas nitroreducens
Other publictions for EC 1.14.13.44
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Synonyms
Temperature Optimum [°C]
Temperature Range [°C]
Temperature Stability [°C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [°C] (protein specific)
Temperature Range [°C] (protein specific)
Temperature Stability [°C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
744407
Kanteev
A crystal structure of 2-hydr ...
Pseudomonas nitroreducens
Biochim. Biophys. Acta
1854
1906-1913
2015
-
-
1
1
6
-
-
12
-
-
-
-
-
5
-
-
1
-
-
-
-
-
2
-
2
-
-
-
12
-
-
-
1
-
-
-
-
-
1
1
1
6
-
-
-
-
12
-
-
-
-
-
-
-
1
-
-
-
-
2
-
-
-
-
12
-
-
-
-
-
-
-
-
12
12
744702
Jensen
Structures of the Apo and FAD ...
Pseudomonas nitroreducens
ChemBioChem
16
968-976
2015
-
-
1
1
-
-
-
4
-
-
-
-
-
3
-
-
1
-
-
-
-
-
1
1
1
-
-
-
-
1
-
-
1
-
-
-
-
-
1
1
1
-
-
-
-
-
4
-
-
-
-
-
-
-
1
-
-
-
-
1
1
-
-
-
-
1
-
-
-
-
-
-
-
-
-
657455
Meyer
Crystallization and preliminar ...
Pseudomonas nitroreducens
Acta Crystallogr. Sect. D
59
741-743
2003
-
-
1
1
-
-
3
-
-
-
1
1
-
7
-
-
1
-
-
-
-
-
2
1
1
-
-
-
-
-
-
-
2
-
-
-
-
-
1
2
1
-
-
-
3
-
-
-
-
1
1
-
-
-
1
-
-
-
-
2
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
438847
Meyer
Changing the substrate reactiv ...
Pseudomonas nitroreducens, Pseudomonas nitroreducens HBP1
J. Biol. Chem.
277
5575-5582
2002
-
-
-
-
2
-
-
1
-
-
-
-
-
10
-
-
-
-
-
-
-
-
10
-
-
-
-
-
4
-
-
-
2
-
-
-
-
-
-
2
-
2
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
10
-
-
-
-
4
-
-
-
-
-
-
-
-
-
-
438848
Meyer
Hydroxylation of indole by lab ...
Pseudomonas nitroreducens, Pseudomonas nitroreducens HBP1
J. Biol. Chem.
277
34161-34167
2002
-
-
-
-
1
-
-
-
-
-
-
-
-
10
-
-
-
-
-
-
-
-
6
-
-
-
-
-
4
-
-
-
1
-
-
-
-
-
-
1
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
6
-
-
-
-
4
-
-
-
-
-
-
-
-
-
-
438849
Suske
Catalytic mechanism of 2-hydro ...
Pseudomonas nitroreducens, Pseudomonas nitroreducens HBP1
J. Biol. Chem.
274
33355-33365
1999
-
-
-
-
-
-
-
2
-
-
-
-
-
10
-
-
1
1
-
-
-
-
8
-
-
-
-
-
1
-
-
-
2
-
-
-
-
-
-
2
-
-
-
-
-
-
2
-
-
-
-
-
-
-
1
-
-
-
-
8
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
658323
Held
An integrated process for the ...
Escherichia coli, Escherichia coli JM101
Biotechnol. Bioeng.
62
641-648
1999
-
1
-
-
-
-
-
-
-
-
-
-
-
12
-
-
-
-
-
-
-
-
2
-
-
1
-
-
-
-
-
-
1
-
-
-
-
1
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
438850
Suske
Purification and characterizat ...
Pseudomonas nitroreducens, Pseudomonas nitroreducens HBP1
J. Biol. Chem.
272
24257-24265
1997
-
-
-
-
-
-
7
10
-
-
2
2
-
16
-
-
1
-
-
-
1
1
18
1
-
-
-
-
11
1
1
-
3
1
-
-
-
-
-
3
-
-
-
-
7
1
10
-
-
2
2
-
-
-
1
-
-
1
1
18
1
-
-
-
11
1
1
-
-
-
-
-
-
-
-