Information on EC 1.3.1.86 - crotonyl-CoA reductase

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY hide
1.3.1.86
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RECOMMENDED NAME
GeneOntology No.
crotonyl-CoA reductase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
butanoyl-CoA + NADP+ = (E)-but-2-enoyl-CoA + NADPH + H+
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
carboxylation
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reductive carboxylation
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Butanoate metabolism
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Biosynthesis of antibiotics
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SYSTEMATIC NAME
IUBMB Comments
butanoyl-CoA:NADP+ 2,3-oxidoreductase
Catalyses the reaction in the reverse direction. This enzyme from Streptomyces collinus is specific for (E)-but-2-enoyl-CoA, and is proposed to provide butanoyl-CoA as a starter unit for straight-chain fatty acid biosynthesis.
CAS REGISTRY NUMBER
COMMENTARY hide
37251-07-3
cf. EC 1.3.1.8
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Peptoclostridium difficile
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-
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
metabolism
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(E)-but-2-enoyl-CoA + NADPH + H+
butanoyl-CoA + NADP+
show the reaction diagram
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?
crotonyl-CoA + NADH + ferredoxin
butyryl-CoA + ?
show the reaction diagram
Peptoclostridium difficile
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-
-
-
?
crotonyl-CoA + NADPH + H+
butanoyl-CoA + NADP+
show the reaction diagram
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the enzyme exhibits a high substrate specificity for crotonyl-CoA
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?
crotonyl-CoA + NADPH + H+
butyryl-CoA + NADP+
show the reaction diagram
trans-crotonyl-CoA + NADPH + H+
butyryl-CoA + NADP+
show the reaction diagram
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the crotonyl-CoA reductase reaction requires NADPH as electron donor
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?
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
FAD
Peptoclostridium difficile
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in the absence of ferredoxin, a remarkable stimulation of crotonyl-CoA reduction by FAD is observed
Ferredoxin
Peptoclostridium difficile
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butyryl-CoA dehydrogenase from C. difficile belongs to the subfamily of bifurcating enzymes capable of coupling the exergonic reduction of crotonyl-CoA by NAD Hwith the endergonic reduction of ferredoxin by NADH
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NADH
Peptoclostridium difficile
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NADPH
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
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at high concentrations beta-mercaptoethanol is inhibitory
2-methylcrotonyl-CoA
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CoA-activated enzyme shows competitive inhibition with the substrate analog 2-methylcrotonyl-CoA
4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole
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extremely potent inhibitor
5,5'-dithiobis(2-nitrobenzoic acid)
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extremely potent inhibitor
acetyl-CoA
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competitive inhibitor
ammonium sulfate
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enzyme activity is inhibited by ammonium sulfatehowever, this inhibition is overcome by addition of 10 mM guanidine
arachidoyl-CoA
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86% residual activity at 0.1 mM
Butyryl-CoA
Ca2+
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complete inhibition at 1 mM
Co2+
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complete inhibition at 1 mM
dithiothreitol
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at high concentrations dithiothreitol is inhibitory
iodoacetamide
iodoacetate
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isomyristoyl-CoA
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78% residual activity at 0.1 mM
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isopalmitoyl-CoA
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95% residual activity at 0.1 mM
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malonyl-CoA
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noncompetitive inhibitor
Mg2+
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30% inhibition at 1 mM
Mn2+
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complete inhibition at 1 mM
myristoyl-CoA
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36% residual activity at 0.1 mM
N-ethylmaleimide
NADPH
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concentrations of NADPH above 0.2 mM lead to inhibition of enzyme activity
p-chloromercuribenzoate
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a 30-min incubation of crotonyl-CoA reductase with p-chloromercuribenzoate at 0.008 mM leads to approximately 8.5% inhibition of enzyme activity
palmitoyl-CoA
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24% residual activity at 0.1 mM
stearoyl-CoA
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92% residual activity at 0.1 mM
Zn2+
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55% inhibition at 1 mM
additional information
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no significant inhibition of the activity of crotonyl-CoA reductase is observed upon addition of either flavin adenine dinucleotide (0.018 and 0.072 mM) or flavin mononucleotide (0.013 and 0.130 mM)
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
CoA
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free CoA is a strong activator of the reductase reaction
L-arabinose
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about 2fold CCR activity is obtained by the addition of 0.1% (w/v) of L-arabinose
additional information
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no significant activation of the activity of crotonyl-CoA reductase is observed upon addition of either flavin adenine dinucleotide (0.018 and 0.072 mM) or flavin mononucleotide (0.013 and 0.130 mM)
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.01
Butyryl-CoA
Peptoclostridium difficile
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pH 7.5, temperature not specified in the publication
0.0025 - 0.018
crotonyl-CoA
0.002
Ferredoxin
Peptoclostridium difficile
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pH 7.5, temperature not specified in the publication
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0.145
NADH
Peptoclostridium difficile
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pH 7.5, temperature not specified in the publication
0.00364 - 0.015
NADPH
0.0033
trans-crotonyl-CoA
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in 200 mM potassium phosphate buffer, pH 6.8, at 26C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2 - 8
Butyryl-CoA
Peptoclostridium difficile
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pH 7.5, temperature not specified in the publication
19
crotonyl-CoA
Peptoclostridium difficile
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pH 7.5, temperature not specified in the publication
37
Ferredoxin
Peptoclostridium difficile
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pH 7.5, temperature not specified in the publication
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3 - 6
NADH
Peptoclostridium difficile
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pH 7.5, temperature not specified in the publication
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2800
Butyryl-CoA
Peptoclostridium difficile
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pH 7.5, temperature not specified in the publication
270
7600
crotonyl-CoA
Peptoclostridium difficile
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pH 7.5, temperature not specified in the publication
406
18500
Ferredoxin
Peptoclostridium difficile
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pH 7.5, temperature not specified in the publication
169
250
NADH
Peptoclostridium difficile
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pH 7.5, temperature not specified in the publication
8
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0029
2-methylcrotonyl-CoA
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pH and temperature not specified in the publication
0.006
acetyl-CoA
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pH and temperature not specified in the publication
0.005 - 0.9
Butyryl-CoA
0.5
isomyristoyl-CoA
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in 50 mM Tris/HCI pH 6.5 and 10% (v/v) glycerol, at 30C
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0.4
isopalmitoyl-CoA
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Ki above 0.4 mM, in 50 mM Tris/HCI pH 6.5 and 10% (v/v) glycerol, at 30C
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0.021
malonyl-CoA
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pH and temperature not specified in the publication
0.017
myristoyl-CoA
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in 50 mM Tris/HCI pH 6.5 and 10% (v/v) glycerol, at 30C
0.63
NADP+
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in 50 mM Tris/HCI pH 6.5 and 10% (v/v) glycerol, at 30C
0.0095
palmitoyl-CoA
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in 50 mM Tris/HCI pH 6.5 and 10% (v/v) glycerol, at 30C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0008
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native enzyme, pH and temperature not specified in the publication
0.00091
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native enzyme from crude extract, pH 7.5 at 30C
0.0061
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crude extract, pH 6.8, at 26C
0.0156
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recombinant enzyme, pH and temperature not specified in the publication
0.0501
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after 11.2fold purification, pH 6.8, at 26C
0.333
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recombinant enzyme from crude extract, pH 7.5 at 30C
2.889
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native enzyme after 3068fold purification, pH 7.5 at 30C
3.316
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recombinant enzyme after 10fold purification, pH 7.5 at 30C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 8.5
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7.5
Peptoclostridium difficile
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assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
85000
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native protein, gel filtration
110000
Peptoclostridium difficile
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
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2 * 48000, SDS-PAGE; 2 * 49400, calculated from amino acid sequence
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40
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at 40C the enzyme retains 47% of its activity after 30 min
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
the crotonyl-CoA reductase activity is influenced by preincubation. 0.25 M Tris-HCl buffer diminishes the crotonyl-CoA reductase activity by approximately 30%
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate precipitation, calcium phosphate gel treatment, alumina gel Cgamma extract filtration, Sephadex G-200 gel filtration
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ammonium sulfate precipitation, DEAE-cellulose column chromatography, phenyl-Sepharose column chromatography, Mono Q column chromatography, Sephadex G-100 gel filtration, and phenyl-Superose gel filtration
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli
Peptoclostridium difficile
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expressed in Escherichia coli BL21 (DE3)/pZYB3 cells
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expressed in Ralstonia eutropha strain PHB-4
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expressed in Streptomyces cinnamonensis strain L1
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expressed in Streptomyces erythraea strain EAT4
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
the addition of 1.0 g/l L-lysine induces expression of the ccr gene in Streptomyces cinnamonensis strain C730.1 in chemically defined medium containing mainly D-glucose (12 g/l), L-tyrosine (3.3 g/l), and either L-valine (6.7 g/l) or L-leucine (7.5 g/l) as carbon and nitrogen sources
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