Cloned (Comment) | Organism |
---|---|
gene hbpA, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Pseudomonas nitroreducens |
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 | 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 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 | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
2-hydroxybiphenyl + NADH + H+ + O2 | Pseudomonas nitroreducens | - |
2,3-dihydroxybiphenyl + NAD+ + H2O | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas nitroreducens | O06647 | Pseudomonas azelaica | - |
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 | 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 | Comment | Organism |
---|---|---|
2-hydroxybiphenyl 3-monooxygenase | - |
Pseudomonas nitroreducens |
HBP1 | - |
Pseudomonas nitroreducens |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Pseudomonas nitroreducens |
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 Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Pseudomonas nitroreducens |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FAD | - |
Pseudomonas nitroreducens | |
NADH | - |
Pseudomonas nitroreducens |
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 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 |