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Literature summary for 1.14.14.9 extracted from

  • Galan, B.; Diaz, E.; Prieto, M.A.; Garcia, J.L.
    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.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli DH1 and TG1 Escherichia coli

General Stability

General Stability Organism
HpaC consists of low stability Escherichia coli

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0021
-
FMN
-
Escherichia coli
0.0026
-
riboflavin
-
Escherichia coli
0.0031
-
FAD
-
Escherichia coli
0.04
-
NADH
-
Escherichia coli

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
18680
-
x * 58781 flavoprotein HpaA, x * 18680, coupling protein, estimation from gene sequence Klebsiella pneumoniae
58781
-
x * 58781 flavoprotein HpaA, x * 18680, coupling protein, estimation from gene sequence Klebsiella pneumoniae

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
4-hydroxyphenylacetate + NADH + O2 Klebsiella pneumoniae
-
3,4-dihydroxyphenylacetate + NAD+ + H2O
-
?
4-hydroxyphenylacetate + NADH + O2 Escherichia coli initial reaction in the degradation of 4-hydroxyphenylacetate 3,4-dihydroxyphenylacetate + NAD+ + H2O
-
?

Organism

Organism UniProt Comment Textmining
Escherichia coli
-
strain W, ATCC 11105 derived from ATCC 9637
-
Klebsiella pneumoniae
-
-
-

Purification (Commentary)

Purification (Comment) Organism
ammonium sulfate precipitation, ion-exchange, gel filtration. Affinity chromatography for the expressed cholin-binding domain containing HpaB protein Escherichia coli

Reaction

Reaction Comment Organism Reaction ID
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

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
0.14
-
activity of HpaB protein in presence of NADH and HpaC protein Escherichia coli
0.46
-
-
Escherichia coli

Storage Stability

Storage Stability Organism
-20°C, HpaC, small protein, very stable, no significant loss of activity during 2 months Escherichia coli

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3-hydroxyphenylacetate + FMNH2 + O2
-
Escherichia coli 3,4-hydroxyphenylacetate + FMN + H2O
-
?
4-hydroxyphenylacetate + NADH + O2
-
Escherichia coli 3,4-dihydroxyphenylacetate + NAD+ + H2O
-
?
4-hydroxyphenylacetate + NADH + O2
-
Klebsiella pneumoniae 3,4-dihydroxyphenylacetate + NAD+ + H2O
-
?
4-hydroxyphenylacetate + NADH + O2 initial reaction in the degradation of 4-hydroxyphenylacetate Escherichia coli 3,4-dihydroxyphenylacetate + NAD+ + H2O
-
?
additional information
-
Klebsiella pneumoniae ?
-
?
additional information hydroxylates phenol derivatives Escherichia coli ?
-
?
phenol + ? for HpaB Escherichia coli catechol + ?
-
?
riboflavin + NADH + H+
-
Escherichia coli reduced riboflavin + NAD+
-
r

Subunits

Subunits Comment Organism
?
-
Escherichia coli
? x * 58781 flavoprotein HpaA, x * 18680, coupling protein, estimation from gene sequence Klebsiella pneumoniae

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
22
-
assay at Escherichia coli

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7
-
optimal for HpaC protein Escherichia coli
7.8
-
assay at Escherichia coli

pH Range

pH Minimum pH Maximum Comment Organism
5 8 more than 80% of activity Escherichia coli

Cofactor

Cofactor Comment Organism Structure
FAD stimulates Escherichia coli
FAD stimulates Klebsiella pneumoniae
FMN used as substrate by HpaC protein Escherichia coli
NADH
-
Klebsiella pneumoniae
NADH electron donor Escherichia coli
riboflavin used as substrate by HpaC protein Escherichia coli