EC Number | Cloned (Comment) | Organism |
---|---|---|
1.5.1.36 | overproduction of the small HpaC component in Escherichia coli K-12 | Escherichia coli |
1.5.1.36 | overproduction of the small HpaC component of the 4-hydroxyphenylacetate 3-monooxygenase in Escherichia coli K-12 cells | Escherichia coli W |
1.14.14.9 | expressed in Escherichia coli DH1 and TG1 | Escherichia coli |
EC Number | General Stability | Organism |
---|---|---|
1.14.14.9 | HpaC consists of low stability | Escherichia coli |
EC Number | KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|---|
1.5.1.36 | 0.0021 | - |
FMN | pH and temperature not specified in the publication | Escherichia coli | |
1.5.1.36 | 0.0021 | - |
FMN | pH 7.8, 22°C, the value is obtained with NADH as the electron donor | Escherichia coli W | |
1.5.1.36 | 0.0026 | - |
riboflavin | pH and temperature not specified in the publication | Escherichia coli | |
1.5.1.36 | 0.0026 | - |
riboflavin | pH 7.8, 22°C, the value is obtained with NADH as the electron donor | Escherichia coli W | |
1.5.1.36 | 0.0031 | - |
FAD | pH and temperature not specified in the publication | Escherichia coli | |
1.5.1.36 | 0.0031 | - |
FAD | pH 7.8, 22°C, the value is obtained with NADH as the electron donor | Escherichia coli W | |
1.5.1.36 | 0.04 | - |
NADH | pH and temperature not specified in the publication | Escherichia coli | |
1.5.1.36 | 0.04 | - |
NADH | pH 7.8, 22°C, the value is obtained with FMN as electron acceptor | Escherichia coli W | |
1.14.14.9 | 0.0021 | - |
FMN | - |
Escherichia coli | |
1.14.14.9 | 0.0026 | - |
riboflavin | - |
Escherichia coli | |
1.14.14.9 | 0.0031 | - |
FAD | - |
Escherichia coli | |
1.14.14.9 | 0.04 | - |
NADH | - |
Escherichia coli |
EC Number | Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|---|
1.5.1.36 | 18522 | - |
2 * 18522, small component of the 4-hydroxyphenylacetate 3-monooxygenase, calculated from sequence | Escherichia coli W |
1.5.1.36 | 18600 | - |
2 * 18600, HpaC, the recombinant small component of the 4-hydroxyphenylacetate 3-monooxygenase is a homodimer, calculated from sequence | Escherichia coli |
1.5.1.36 | 20000 | - |
2 * 20000, SDS-PAGE | Escherichia coli W |
1.5.1.36 | 20000 | - |
2 * 20000, HpaC, the recombinant small component of the 4-hydroxyphenylacetate 3-monooxygenase is a homodimer, SDS-PAGE | Escherichia coli |
1.14.14.9 | 18680 | - |
x * 58781 flavoprotein HpaA, x * 18680, coupling protein, estimation from gene sequence | Klebsiella pneumoniae |
1.14.14.9 | 58781 | - |
x * 58781 flavoprotein HpaA, x * 18680, coupling protein, estimation from gene sequence | Klebsiella pneumoniae |
EC Number | Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
1.5.1.36 | FAD + NADH + H+ | Escherichia coli | HpaC, the small reductase component of the 4-hydroxyphenylacetate 3-monooxygenase. The reductase (HpaC) and the oxygenase component (HpaB) of the monooxygenase are encoded by two different genes. The reductase component uses NADH to catalyze the reduction of a flavin that diffuses to the oxygenase component for oxidation of the substrate by molecular oxygen. HpaC that is recombinantly overproduced in Escherichia coli K12 catalyzes the reduction of free flavins by NADH in preference to NADPH | FADH2 + NAD+ | - |
? | |
1.5.1.36 | FMN + NADH + H+ | Escherichia coli | HpaC, the small reductase component of the 4-hydroxyphenylacetate 3-monooxygenase. The reductase (HpaC) and the oxygenase component (HpaB) of the monooxygenase are encoded by two different genes. The reductase component uses NADH to catalyze the reduction of a flavin that diffuses to the oxygenase component for oxidation of the substrate by molecular oxygen. HpaC that is recombinantly overproduced in Escherichia coli K12 catalyzes the reduction of free flavins by NADH in preference to NADPH | FMNH2 + NAD+ | - |
? | |
1.5.1.36 | riboflavin + NADH + H+ | Escherichia coli | HpaC, the small reductase component of the 4-hydroxyphenylacetate 3-monooxygenase. The reductase (HpaC) and the oxygenase component (HpaB) of the monooxygenase are encoded by two different genes. The reductase component uses NADH to catalyze the reduction of a flavin that diffuses to the oxygenase component for oxidation of the substrate by molecular oxygen. HpaC that is recombinantly overproduced in Escherichia coli K12 catalyzes the reduction of free flavins by NADH in preference to NADPH | reduced riboflavin + NAD+ | - |
? | |
1.14.14.9 | 4-hydroxyphenylacetate + NADH + O2 | Klebsiella pneumoniae | - |
3,4-dihydroxyphenylacetate + NAD+ + H2O | - |
? | |
1.14.14.9 | 4-hydroxyphenylacetate + NADH + O2 | Escherichia coli | initial reaction in the degradation of 4-hydroxyphenylacetate | 3,4-dihydroxyphenylacetate + NAD+ + H2O | - |
? |
EC Number | Organism | UniProt | Comment | Textmining |
---|---|---|---|---|
1.5.1.36 | Escherichia coli | - |
- |
- |
1.5.1.36 | Escherichia coli W | Q57501 | - |
- |
1.14.14.9 | Escherichia coli | - |
strain W, ATCC 11105 derived from ATCC 9637 | - |
1.14.14.9 | Klebsiella pneumoniae | - |
- |
- |
EC Number | Purification (Comment) | Organism |
---|---|---|
1.5.1.36 | - |
Escherichia coli |
1.5.1.36 | recombinant enzyme produced in Escherichia coli K-12 cells | Escherichia coli W |
1.14.14.9 | ammonium sulfate precipitation, ion-exchange, gel filtration. Affinity chromatography for the expressed cholin-binding domain containing HpaB protein | Escherichia coli |
EC Number | Reaction | Comment | Organism | Reaction ID |
---|---|---|---|---|
1.14.14.9 | 4-hydroxyphenylacetate + FADH2 + O2 = 3,4-dihydroxyphenylacetate + FAD + H2O | member of novel two-component flavin-diffusible monooxygenase family, both components required for hydroxylation. The genes are located on the same operon. Physical interaction between HpaB and HpaC is not required, enhancement of activity by direct interaction is not excluded. Contains reductase component | Escherichia coli |
EC Number | Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|---|
1.14.14.9 | 0.14 | - |
activity of HpaB protein in presence of NADH and HpaC protein | Escherichia coli |
1.14.14.9 | 0.46 | - |
- |
Escherichia coli |
EC Number | Storage Stability | Organism |
---|---|---|
1.5.1.36 | -20°C, 2 months, no significant loss of activity | Escherichia coli |
1.5.1.36 | -20°C, no significant loss of activity is observed during 2 months of storage at this temperature | Escherichia coli W |
1.14.14.9 | -20°C, HpaC, small protein, very stable, no significant loss of activity during 2 months | Escherichia coli |
EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
1.5.1.36 | FAD + NADH + H+ | HpaC, the small reductase component of the 4-hydroxyphenylacetate 3-monooxygenase. The reductase (HpaC) and the oxygenase component (HpaB) of the monooxygenase are encoded by two different genes. The reductase component uses NADH to catalyze the reduction of a flavin that diffuses to the oxygenase component for oxidation of the substrate by molecular oxygen. HpaC that is recombinantly overproduced in Escherichia coli K12 catalyzes the reduction of free flavins by NADH in preference to NADPH | Escherichia coli | FADH2 + NAD+ | - |
? | |
1.5.1.36 | FAD + NADH + H+ | although the HpaC enzyme can also use NADPH as a substrate, its specific activities on FMN, FAD, and riboflavin are more than 2 orders of magnitude lower than those observed in the presence of NADH. Vmax/Km is 37% compared to the value for the reaction of FMN + NADH | Escherichia coli W | FADH2 + NAD+ | - |
? | |
1.5.1.36 | FMN + NADH + H+ | HpaC, the small reductase component of the 4-hydroxyphenylacetate 3-monooxygenase. The reductase (HpaC) and the oxygenase component (HpaB) of the monooxygenase are encoded by two different genes. The reductase component uses NADH to catalyze the reduction of a flavin that diffuses to the oxygenase component for oxidation of the substrate by molecular oxygen. HpaC that is recombinantly overproduced in Escherichia coli K12 catalyzes the reduction of free flavins by NADH in preference to NADPH | Escherichia coli | FMNH2 + NAD+ | - |
? | |
1.5.1.36 | FMN + NADH + H+ | the most effective substrates are NADH and FMN. When FMN is added in a 200fold molar excess of the HpaC protein, it becomes completely reduced, suggesting that the flavin dissociates from the protein and behaves as a true substrate rather than as a tightly bound cofactor. Although the HpaC enzyme can also use NADPH as a substrate, its specific activities on FMN, FAD, and riboflavin are more than 2 orders of magnitude lower than those observed in the presence of NADH | Escherichia coli W | FMNH2 + NAD+ | - |
? | |
1.5.1.36 | riboflavin + NADH + H+ | HpaC, the small reductase component of the 4-hydroxyphenylacetate 3-monooxygenase. The reductase (HpaC) and the oxygenase component (HpaB) of the monooxygenase are encoded by two different genes. The reductase component uses NADH to catalyze the reduction of a flavin that diffuses to the oxygenase component for oxidation of the substrate by molecular oxygen. HpaC that is recombinantly overproduced in Escherichia coli K12 catalyzes the reduction of free flavins by NADH in preference to NADPH | Escherichia coli | reduced riboflavin + NAD+ | - |
? | |
1.5.1.36 | riboflavin + NADH + H+ | although the HpaC enzyme can also use NADPH as a substrate, its specific activities on FMN, FAD, and riboflavin are more than 2 orders of magnitude lower than those observed in the presence of NADH. Vmax/Km is 70% compared to the value for the reaction of FMN + NADH | Escherichia coli W | reduced riboflavin + NAD+ | - |
? | |
1.14.14.9 | 3-hydroxyphenylacetate + FMNH2 + O2 | - |
Escherichia coli | 3,4-hydroxyphenylacetate + FMN + H2O | - |
? | |
1.14.14.9 | 4-hydroxyphenylacetate + NADH + O2 | - |
Escherichia coli | 3,4-dihydroxyphenylacetate + NAD+ + H2O | - |
? | |
1.14.14.9 | 4-hydroxyphenylacetate + NADH + O2 | - |
Klebsiella pneumoniae | 3,4-dihydroxyphenylacetate + NAD+ + H2O | - |
? | |
1.14.14.9 | 4-hydroxyphenylacetate + NADH + O2 | initial reaction in the degradation of 4-hydroxyphenylacetate | Escherichia coli | 3,4-dihydroxyphenylacetate + NAD+ + H2O | - |
? | |
1.14.14.9 | additional information | - |
Klebsiella pneumoniae | ? | - |
? | |
1.14.14.9 | additional information | hydroxylates phenol derivatives | Escherichia coli | ? | - |
? | |
1.14.14.9 | phenol + ? | for HpaB | Escherichia coli | catechol + ? | - |
? | |
1.14.14.9 | riboflavin + NADH + H+ | - |
Escherichia coli | reduced riboflavin + NAD+ | - |
r |
EC Number | Subunits | Comment | Organism |
---|---|---|---|
1.5.1.36 | dimer | 2 * 18600, HpaC, the recombinant small component of the 4-hydroxyphenylacetate 3-monooxygenase is a homodimer, calculated from sequence | Escherichia coli |
1.5.1.36 | dimer | 2 * 20000, HpaC, the recombinant small component of the 4-hydroxyphenylacetate 3-monooxygenase is a homodimer, SDS-PAGE | Escherichia coli |
1.5.1.36 | homodimer | 2 * 20000, SDS-PAGE | Escherichia coli W |
1.5.1.36 | homodimer | 2 * 18522, small component of the 4-hydroxyphenylacetate 3-monooxygenase, calculated from sequence | Escherichia coli W |
1.5.1.36 | More | overproduction of the small HpaC component in Escherichia coli K-12 cells facilitates the purification of the protein, which is a homodimer that catalyzes the reduction of free flavins by NADH in preference to NADPH | Escherichia coli W |
1.14.14.9 | ? | - |
Escherichia coli |
1.14.14.9 | ? | x * 58781 flavoprotein HpaA, x * 18680, coupling protein, estimation from gene sequence | Klebsiella pneumoniae |
EC Number | Synonyms | Comment | Organism |
---|---|---|---|
1.5.1.36 | flavin:NADH oxidoreductase | - |
Escherichia coli |
1.5.1.36 | flavin:NADH oxidoreductase | - |
Escherichia coli W |
1.5.1.36 | HpaC | small component of the 4-hydroxyphenylacetate 3-monooxygenase | Escherichia coli |
1.5.1.36 | HpaC | small component of the 4-hydroxyphenylacetate 3-monooxygenase in Escherichia coli K-12 cells | Escherichia coli W |
1.5.1.36 | NADH-dependent flavin reductase | - |
Escherichia coli |
EC Number | Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|---|
1.5.1.36 | 22 | - |
assay at | Escherichia coli W |
1.14.14.9 | 22 | - |
assay at | Escherichia coli |
EC Number | pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|---|
1.5.1.36 | 7 | - |
- |
Escherichia coli W |
1.5.1.36 | 7.8 | - |
assay at | Escherichia coli W |
1.14.14.9 | 7 | - |
optimal for HpaC protein | Escherichia coli |
1.14.14.9 | 7.8 | - |
assay at | Escherichia coli |
EC Number | pH Minimum | pH Maximum | Comment | Organism |
---|---|---|---|---|
1.5.1.36 | 6.5 | 8 | the enzyme maintains more than 80% of activity between pH values of 6.5 to 8 | Escherichia coli W |
1.14.14.9 | 5 | 8 | more than 80% of activity | Escherichia coli |
EC Number | Cofactor | Comment | Organism | Structure |
---|---|---|---|---|
1.5.1.36 | NADH | HpaC, the small component of the 4-hydroxyphenylacetate 3-monooxygenase, is recombinantly overproduced in Escherichia coli K12 and catalyzes the reduction of free flavins by NADH in preference to NADPH | Escherichia coli | |
1.5.1.36 | NADH | although the HpaC enzyme can also use NADPH as a substrate, its specific activities on FMN, FAD, and riboflavin are more than 2 orders of magnitude lower than those observed in the presence of NADH | Escherichia coli W | |
1.5.1.36 | NADPH | although the HpaC enzyme can also use NADPH as a substrate, its specific activities on FMN, FAD, and riboflavin are more than 2 orders of magnitude lower than those observed in the presence of NADH | Escherichia coli W | |
1.14.14.9 | FAD | stimulates | Escherichia coli | |
1.14.14.9 | FAD | stimulates | Klebsiella pneumoniae | |
1.14.14.9 | FMN | used as substrate by HpaC protein | Escherichia coli | |
1.14.14.9 | NADH | - |
Klebsiella pneumoniae | |
1.14.14.9 | NADH | electron donor | Escherichia coli | |
1.14.14.9 | riboflavin | used as substrate by HpaC protein | Escherichia coli |
EC Number | General Information | Comment | Organism |
---|---|---|---|
1.5.1.36 | physiological function | the 4-hydroxyphenylacetate 3-monooxygenase from Escherichia coli W is a two-component enzyme encoded by the hpaB and hpaC genes and catalyzes the initial reaction in the degradation of 4-hydroxyphenylacetate, i.e., the introduction of a second hydroxyl group into the benzene nucleus at a position ortho to the existing hydroxyl group, giving rise to 3,4-dihydroxyphenylacetate | Escherichia coli W |
EC Number | kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|---|
1.5.1.36 | additional information | - |
additional information | V(max)/Km-values in 1/min*mg: 33.3 for FMN (with NADH as electron donor), 22.7 for riboflavin (with NADH as electron donor), 12.3 for FAD (with NADH as electron donor) | Escherichia coli W |