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Literature summary for 1.5.1.36 extracted from
- Functional analysis of the small component of the 4-hydroxyphenylacetate 3-monooxygenase of Escherichia coli W: a prototype of a new flavin:NAD(P)H reductase subfamily (2000), J. Bacteriol., 182, 627-636.
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| overproduction of the small HpaC component in Escherichia coli K-12 | Escherichia coli |
| overproduction of the small HpaC component of the 4-hydroxyphenylacetate 3-monooxygenase in Escherichia coli K-12 cells | Escherichia coli W |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.0021 | - |
FMN | pH and temperature not specified in the publication | Escherichia coli |  |
| 0.0021 | - |
FMN | pH 7.8, 22°C, the value is obtained with NADH as the electron donor | Escherichia coli W | |
| 0.0026 | - |
riboflavin | pH and temperature not specified in the publication | Escherichia coli | |
| 0.0026 | - |
riboflavin | pH 7.8, 22°C, the value is obtained with NADH as the electron donor | Escherichia coli W | |
| 0.0031 | - |
FAD | pH and temperature not specified in the publication | Escherichia coli | |
| 0.0031 | - |
FAD | pH 7.8, 22°C, the value is obtained with NADH as the electron donor | Escherichia coli W | |
| 0.04 | - |
NADH | pH and temperature not specified in the publication | Escherichia coli | |
| 0.04 | - |
NADH | pH 7.8, 22°C, the value is obtained with FMN as electron acceptor | Escherichia coli W | |
Molecular Weight [Da]
| Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
|---|---|---|---|
| 18522 | - |
2 * 18522, small component of the 4-hydroxyphenylacetate 3-monooxygenase, calculated from sequence | Escherichia coli W |
| 18600 | - |
2 * 18600, HpaC, the recombinant small component of the 4-hydroxyphenylacetate 3-monooxygenase is a homodimer, calculated from sequence | Escherichia coli |
| 20000 | - |
2 * 20000, SDS-PAGE | Escherichia coli W |
| 20000 | - |
2 * 20000, HpaC, the recombinant small component of the 4-hydroxyphenylacetate 3-monooxygenase is a homodimer, SDS-PAGE | Escherichia coli |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 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+ | - |
? | |
| 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+ | - |
? | |
| 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+ | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Escherichia coli | - |
- |
- |
| Escherichia coli W | Q57501 | - |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
- |
Escherichia coli |
| recombinant enzyme produced in Escherichia coli K-12 cells | Escherichia coli W |
Storage Stability
| Storage Stability | Organism |
|---|---|
| -20°C, 2 months, no significant loss of activity | Escherichia coli |
| -20°C, no significant loss of activity is observed during 2 months of storage at this temperature | Escherichia coli W |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 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+ | - |
? | |
| 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+ | - |
? | |
| 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+ | - |
? | |
| 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+ | - |
? | |
| 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+ | - |
? | |
| 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+ | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| dimer | 2 * 18600, HpaC, the recombinant small component of the 4-hydroxyphenylacetate 3-monooxygenase is a homodimer, calculated from sequence | Escherichia coli |
| dimer | 2 * 20000, HpaC, the recombinant small component of the 4-hydroxyphenylacetate 3-monooxygenase is a homodimer, SDS-PAGE | Escherichia coli |
| homodimer | 2 * 20000, SDS-PAGE | Escherichia coli W |
| homodimer | 2 * 18522, small component of the 4-hydroxyphenylacetate 3-monooxygenase, calculated from sequence | Escherichia coli W |
| 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 |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| flavin:NADH oxidoreductase | - |
Escherichia coli |
| flavin:NADH oxidoreductase | - |
Escherichia coli W |
| HpaC | small component of the 4-hydroxyphenylacetate 3-monooxygenase | Escherichia coli |
| HpaC | small component of the 4-hydroxyphenylacetate 3-monooxygenase in Escherichia coli K-12 cells | Escherichia coli W |
| NADH-dependent flavin reductase | - |
Escherichia coli |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 22 | - |
assay at | Escherichia coli W |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 7 | - |
- |
Escherichia coli W |
| 7.8 | - |
assay at | Escherichia coli W |
pH Range
| pH Minimum | pH Maximum | Comment | Organism |
|---|---|---|---|
| 6.5 | 8 | the enzyme maintains more than 80% of activity between pH values of 6.5 to 8 | Escherichia coli W |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| 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 | |
| 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 | |
| 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 | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| 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 |
kcat/KM [mM/s]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 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 |
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