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Literature summary for 1.14.13.44 extracted from

  • Bregman-Cohen, A.; Deri, B.; Maimon, S.; Pazy, Y.; Fishman, A.
    Altering 2-hydroxybiphenyl 3-monooxygenase regioselectivity by protein engineering for the production of a new antioxidant (2018), ChemBioChem, 19, 583-590 .
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
gene hbpA, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Pseudomonas nitroreducens

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinant enzyme HbpA mutant M321A, X-ray diffraction structure determination and anylysis at 2.78 A resolution, molecular replacement and structure modeling Pseudomonas nitroreducens

Protein Variants

Protein Variants Comment Organism
M223A site-directed saturation mutagenesis, the mutant shows reduced activity compared to wild-type enzyme Pseudomonas nitroreducens
M223E site-directed saturation mutagenesis, the mutant shows reduced activity compared to wild-type enzyme Pseudomonas nitroreducens
M223I site-directed saturation mutagenesis, the mutant shows reduced activity compared to wild-type enzyme Pseudomonas nitroreducens
M223K site-directed saturation mutagenesis, the mutant shows reduced activity compared to wild-type enzyme Pseudomonas nitroreducens
M223Q site-directed saturation mutagenesis, the mutant shows reduced activity compared to wild-type enzyme Pseudomonas nitroreducens
M321A site-directed saturation mutagenesis, the mutant variant demonstrates altered regioselectivity by oxidizing 3-hydroxybiphenyl, and thus enabling the production of a distinct antioxidant, 3,4-dihydroxybiphenyl, with similar ferric reducing capacity to the well-studied piceatannol. Mutant enzyme crystal structure analysis and comparison to the wild-type enzyme structure Pseudomonas nitroreducens
M321F site-directed saturation mutagenesis, wild-type HbpA possess pro-S enantioselectivity towards the production of several chiral sulfoxides, whereas the mutant M321F exhibits improved enantioselectivity and increased activity compared to wild-type Pseudomonas nitroreducens
M321L site-directed saturation mutagenesis, the mutant shows reduced activity compared to wild-type enzyme Pseudomonas nitroreducens
M321V site-directed saturation mutagenesis, the mutant shows reduced activity compared to wild-type enzyme Pseudomonas nitroreducens
additional information site-directed saturation mutagenesis of HbpA and library screening, altering 2-hydroxybiphenyl 3-monooxygenase regioselectivity by protein engineering for the production of a new antioxidant. Modulation of the enzyme activity and selectivity via mutation of several residues in the active site pocket Pseudomonas nitroreducens
P320X site-directed mutagenesis, all mutations of Pro320 in the library result in activity loss. Therefore, it appears that a proline at this position is crucial for the catalytic reaction. The short distance of Pro320 from the FAD cofactor implies its involvement in enabling the movement of FAD, which is imperative for HbpA activity. Pro320 is conserved among flavin monooxygenases. In aklavinone-11-hydroxylase (RdmE), para-hydroxybenzoate hydroxylase (pHBH), and phenol hydroxylase (PH), this position next to the FAD is occupied by Pro315, Pro293, and Pro364, respectively Pseudomonas nitroreducens
W97A site-directed saturation mutagenesis, the mutant shows reduced activity compared to wild-type enzyme Pseudomonas nitroreducens
W97Y site-directed saturation mutagenesis, the mutant shows reduced activity compared to wild-type enzyme Pseudomonas nitroreducens

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0018
-
2-Hydroxybiphenyl mutant M321F, pH 7.5, 30°C Pseudomonas nitroreducens
0.0028
-
2-Hydroxybiphenyl mutant M223E, pH 7.5, 30°C Pseudomonas nitroreducens
0.0031
-
2-Hydroxybiphenyl recombinant wild-type enzyme, pH 7.5, 30°C Pseudomonas nitroreducens
0.006
-
2-Hydroxybiphenyl mutant W97A, pH 7.5, 30°C Pseudomonas nitroreducens
0.0065
-
2-Hydroxybiphenyl mutant M223K, pH 7.5, 30°C Pseudomonas nitroreducens
0.0123
-
2-Hydroxybiphenyl mutant M223A, pH 7.5, 30°C Pseudomonas nitroreducens
0.0153
-
2-Hydroxybiphenyl mutant M321V, pH 7.5, 30°C Pseudomonas nitroreducens
0.0156
-
2-Hydroxybiphenyl mutant M321L, pH 7.5, 30°C Pseudomonas nitroreducens
0.0169
-
2-Hydroxybiphenyl mutant W97Y, pH 7.5, 30°C Pseudomonas nitroreducens
0.0236
-
2-Hydroxybiphenyl mutant M223I, pH 7.5, 30°C Pseudomonas nitroreducens
0.0462
-
2-Hydroxybiphenyl mutant M321A, pH 7.5, 30°C Pseudomonas nitroreducens
0.0787
-
2-Hydroxybiphenyl mutant M223Q, pH 7.5, 30°C Pseudomonas nitroreducens

Natural Substrates/ Products (Substrates)

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

Organism

Organism UniProt Comment Textmining
Pseudomonas nitroreducens O06647 Pseudomonas azelaica
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and dialysis Pseudomonas nitroreducens

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2-hydroxybiphenyl + NADH + H+ + O2
-
Pseudomonas nitroreducens 2,3-dihydroxybiphenyl + NAD+ + H2O
-
?
3-hydroxybiphenyl + NADH + H+ + O2 activity of enzyme mutant M321A Pseudomonas nitroreducens 3,4-dihydroxybiphenyl + NAD+ + H2O
-
?
additional information wild-type enzyme HbpA has a broad substrate range and catalyzes the regioselective ortho-hydroxylation of a wide range of 2-substituted phenols to the corresponding catechols. It possess pro-S enantioselectivity towards the production of several chiral sulfoxides, whereas its mutant variant M321F exhibits improved enantioselectivity, while mutant M321A shows altered regioselectivity by oxidizing 3-hydroxybiphenyl, and thus enabling the production of a distinct antioxidant, 3,4-dihydroxybiphenyl. Identification of substrates and products by GC/MS Pseudomonas nitroreducens ?
-
-

Synonyms

Synonyms Comment Organism
2-hydroxybiphenyl 3-monooxygenase
-
Pseudomonas nitroreducens
HBP1
-
Pseudomonas nitroreducens

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
assay at Pseudomonas nitroreducens

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.46
-
2-Hydroxybiphenyl mutant W97Y, pH 7.5, 30°C Pseudomonas nitroreducens
0.48
-
2-Hydroxybiphenyl mutant W97A, pH 7.5, 30°C Pseudomonas nitroreducens
0.73
-
2-Hydroxybiphenyl mutant M223E, pH 7.5, 30°C Pseudomonas nitroreducens
1.24
-
2-Hydroxybiphenyl mutant M223A, pH 7.5, 30°C Pseudomonas nitroreducens
1.83
-
2-Hydroxybiphenyl mutant M223I, pH 7.5, 30°C Pseudomonas nitroreducens
1.97
-
2-Hydroxybiphenyl mutant M223K, pH 7.5, 30°C Pseudomonas nitroreducens
2.26
-
2-Hydroxybiphenyl recombinant wild-type enzyme, pH 7.5, 30°C Pseudomonas nitroreducens
2.75
-
2-Hydroxybiphenyl mutant M321V, pH 7.5, 30°C Pseudomonas nitroreducens
2.79
-
2-Hydroxybiphenyl mutant M321L, pH 7.5, 30°C Pseudomonas nitroreducens
3.01
-
2-Hydroxybiphenyl mutant M321A, pH 7.5, 30°C Pseudomonas nitroreducens
3.94
-
2-Hydroxybiphenyl mutant M321F, pH 7.5, 30°C Pseudomonas nitroreducens
4.54
-
2-Hydroxybiphenyl mutant M223Q, pH 7.5, 30°C Pseudomonas nitroreducens

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
assay at Pseudomonas nitroreducens

Cofactor

Cofactor Comment Organism Structure
FAD
-
Pseudomonas nitroreducens
NADH
-
Pseudomonas nitroreducens

General Information

General Information Comment Organism
additional information residue Trp97 stabilizes the substrate in the active site, residue Met223 is involved in NADH entrance or binding to the active site, and residue Pro320 might facilitate FAD movement. Molecular docking study with wild-type and mutant enzymes and FAD and substrates Pseudomonas nitroreducens
physiological function 2-hydroxybiphenyl 3-monooxygenase is a flavin-containing NADH-dependent aromatic hydroxylase that oxidizes a broad range of 2-substituted phenols Pseudomonas nitroreducens

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
27.2
-
2-Hydroxybiphenyl mutant W97Y, pH 7.5, 30°C Pseudomonas nitroreducens
57.7
-
2-Hydroxybiphenyl mutant M223Q, pH 7.5, 30°C Pseudomonas nitroreducens
65.2
-
2-Hydroxybiphenyl mutant M321A, pH 7.5, 30°C Pseudomonas nitroreducens
77.5
-
2-Hydroxybiphenyl mutant M223I, pH 7.5, 30°C Pseudomonas nitroreducens
80
-
2-Hydroxybiphenyl mutant W97A, pH 7.5, 30°C Pseudomonas nitroreducens
100.8
-
2-Hydroxybiphenyl mutant M223A, pH 7.5, 30°C Pseudomonas nitroreducens
178.9
-
2-Hydroxybiphenyl mutant M321L, pH 7.5, 30°C Pseudomonas nitroreducens
179.7
-
2-Hydroxybiphenyl mutant M321V, pH 7.5, 30°C Pseudomonas nitroreducens
260.7
-
2-Hydroxybiphenyl mutant M223E, pH 7.5, 30°C Pseudomonas nitroreducens
303.1
-
2-Hydroxybiphenyl mutant M223K, pH 7.5, 30°C Pseudomonas nitroreducens
729
-
2-Hydroxybiphenyl recombinant wild-type enzyme, pH 7.5, 30°C Pseudomonas nitroreducens
2188.9
-
2-Hydroxybiphenyl mutant M321F, pH 7.5, 30°C Pseudomonas nitroreducens