Information on EC 1.5.1.36 - flavin reductase (NADH)

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.5.1.36
-
RECOMMENDED NAME
GeneOntology No.
flavin reductase (NADH)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
reduced flavin + NAD+ = flavin + NADH + H+
show the reaction diagram
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-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Metabolic pathways
-
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Riboflavin metabolism
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SYSTEMATIC NAME
IUBMB Comments
flavin:NAD+ oxidoreductase
The enzyme from Escherichia coli W catalyses the reduction of free flavins by NADH. The enzyme has similar affinity to FAD, FMN and riboflavin. Activity with NADPH is more than 2 orders of magnitude lower than activity with NADH.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
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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
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
FAD + NADH + H+
FADH2 + NAD+
show the reaction diagram
FMN + NADH + H+
FMNH2 + NAD+
show the reaction diagram
riboflavin + NADH + H+
reduced riboflavin + NAD+
show the reaction diagram
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
FAD + NADH + H+
FADH2 + NAD+
show the reaction diagram
FMN + NADH + H+
FMNH2 + NAD+
show the reaction diagram
riboflavin + NADH + H+
reduced riboflavin + NAD+
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
FMN
-
each subunit binds one FMN as cofactor
NADPH
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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
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Cr3+
-
slight activation by 1 mM Cr3+
Zn2+
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slight activation by 1 mM Zn2+
additional information
-
the enzyme is not susceptible to Ca2+, Mg2+, Mn2+ and Co2+
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Ag+
-
strong inhibition at 1 mM Ag+
Cu2+
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strong inhibition at 1 mM Cu2+
Hg2+
-
strong inhibition at 1 mM Hg2+
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0031
FAD
0.0021 - 0.0066
FMN
0.015 - 0.0779
NADH
0.0026
riboflavin
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.7
FMN
Photobacterium leiognathi
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at 4C in 50 mM Tris-Cl buffer, pH 8.0
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
additional information
Escherichia coli W
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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)
2
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.6
-
using riboflavin and NADPH as substrates, at pH 7.0 and 55C
0.8
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using FAD and NADPH as substrates, at pH 7.0 and 55C
1.3
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using FMN and NADPH as substrates, at pH 7.0 and 55C
36
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using riboflavin and NADH as substrates, at pH 7.0 and 55C
189
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using FAD and NADH as substrates, at pH 7.0 and 55C
617
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using FMN and NADH as substrates, at pH 7.0 and 55C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.8
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assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 8
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the enzyme maintains more than 80% of activity between pH values of 6.5 to 8
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22
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assay at
PDB
SCOP
CATH
ORGANISM
UNIPROT
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
16989
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2 * 16989, calculated from amino acid sequence
17000
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2 * 17000, SDS-PAGE
18522
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2 * 18522, small component of the 4-hydroxyphenylacetate 3-monooxygenase, calculated from sequence
18600
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2 * 18600, HpaC, the recombinant small component of the 4-hydroxyphenylacetate 3-monooxygenase is a homodimer, calculated from sequence
18679
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2 * 18679, calculated from sequence
19400
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x * 19400, calculated from amino acid sequence
39000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 19400, calculated from amino acid sequence
homodimer
additional information
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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
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45 - 60
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enzyme activity is stable at temperatures below 45C. After incubation at 60C for 30 min, more than 90% of the activity is lost
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 2 months, no significant loss of activity
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-20C, no significant loss of activity is observed during 2 months of storage at this temperature
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate precipitation, butyl-Sepharose column chromatography, Source 30Q column chromatography, FMN-Sepharose 6B affinity column chromatography, and Superdex 75 gel filtration
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Ni2+ column chromatography
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recombinant enzyme produced in Escherichia coli K-12 cells
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) cells
overexpression in Escherichia coli
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overproduction of the small HpaC component in Escherichia coli K-12
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overproduction of the small HpaC component of the 4-hydroxyphenylacetate 3-monooxygenase in Escherichia coli K-12 cells
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