Literature summary for 1.14.13.44 extracted from

Kanteev, M.; Bregman-Cohen, A.; Deri, B.; Shahar, A.; Adir, N.; Fishman, A.
A crystal structure of 2-hydroxybiphenyl 3-monooxygenase with bound substrate provides insights into the enzymatic mechanism (2015), Biochim. Biophys. Acta, 1854, 1906-1913 .

Search for more literature for 1.14.13.44

Cloned(Commentary)

Cloned (Comment)	Organism
expression in Escherichia coli	Pseudomonas nitroreducens

Crystallization (Commentary)

Crystallization (Comment)	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

Crystallization (Comment)

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

Protein Variants

Protein Variants	Comment	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	Comment	Organism
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	Comment	Textmining
Pseudomonas nitroreducens	O06647	-	-

Purification (Commentary)

Purification (Comment)	Organism
-	Pseudomonas nitroreducens

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
2,3-dihydroxybiphenyl + NADH + H+ + O2	-	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	Pseudomonas nitroreducens	2,3-dihydroxybiphenyl + NAD+ + H2O	-	?

Synonyms

Synonyms	Comment	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	Comment	Organism
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	Comment	Organism	Structure
FAD	residue Arg242 is suggested to facilitate FAD movement and reduction	Pseudomonas nitroreducens

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
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