Information on EC 4.2.1.157 - (R)-2-hydroxyisocaproyl-CoA dehydratase

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The expected taxonomic range for this enzyme is: Clostridioides difficile

EC NUMBER
COMMENTARY hide
4.2.1.157
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RECOMMENDED NAME
GeneOntology No.
(R)-2-hydroxyisocaproyl-CoA dehydratase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(R)-2-hydroxy-4-methylpentanoyl-CoA = 4-methylpent-2-enoyl-CoA + H2O
show the reaction diagram
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
leucine degradation IV
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SYSTEMATIC NAME
IUBMB Comments
(R)-2-hydroxy-4-methylpentanoyl-CoA hydro-lyase
The enzyme, isolated from the bacterium Peptoclostridium difficile, is involved in the reductive branch of L-leucine fermentation. It catalyses an alpha/beta-dehydration, which depends on the reductive formation of ketyl radicals on the substrate generated by injection of a single electron from the ATP-dependent activator protein HadI.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(R)-2-hydroxy-4-methylpentanoyl-CoA
4-methylpent-2-enoyl-CoA + H2O
show the reaction diagram
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
(R)-2-hydroxy-4-methylpentanoyl-CoA
4-methylpent-2-enoyl-CoA + H2O
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
[4Fe-4S]-center
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a [4Fe-4S] cluster-containing protein activates 2-hydroxyisocaproyl-CoA dehydratase by an ATP-driven electron transfer. Iron chelation by bathophenanthroline removes the reduced [4Fe-4S] cluster from the activator protein in an ATP-dependent manner. With ADP, no chelation is observed. Chelation of the oxidised [4Fe-4S] cluster occurs faster with ADP than with ATP
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Zn2+
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besides varying amounts of zinc, other metal ions, particularly molybdenum, are not detected in the dehydratase
[4Fe-4S] center
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the crystal structure reveals that the heterodimeric protein contains two [4Fe-4S] clusters at a distance of 12 A, each coordinated by three cysteines and one terminal ligand. The cluster in the alpha-subunit is part of the active site. In the absence of substrate, a water/hydroxide ion acts as the fourth ligand. The substrate replaces this ligand and coordinates the cluster via the carbonyl-oxygen of the thioester group. The cluster in the beta-subunit has a terminal sulfhydryl/sulfido ligand and can act as a reservoir to protect the electron from unwanted side reactions via a recycling mechanism
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
AlF4-
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AlF4 in combination with ADP traps the interaction of the activator protein with the dehydratase by forming a stable complex containing 1.0 mol homodimeric activator, 1.0 mol heterodimeric dehydratase and 1.2 mol ADP. The formation proceeds much slower than the activation but in an almost irreversible manner. The isolated complex is devoid of any activity
bathophenanthroline
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a [4Fe-4S] cluster-containing protein activates 2-hydroxyisocaproyl-CoA dehydratase by an ATP-driven electron transfer. Iron chelation by bathophenanthroline removes the reduced [4Fe-4S] cluster from the activator protein in an ATP-dependent manner. With ADP, no chelation is observed. Chelation of the oxidised [4Fe-4S] cluster occurs faster with ADP than with ATP
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
activator HadI
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[4Fe-4S]-center
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additional information
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a [4Fe-4S] cluster-containing protein activates 2-hydroxyisocaproyl-CoA dehydratase by an ATP-driven electron transfer. Reduction of the activator protein and binding of ATP induce conformational changes necessary to transfer the electron to the dehydratase. Interaction of both proteins promotes ATP hydrolysis
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.05 - 0.08
(R)-2-hydroxy-4-methylpentanoyl-CoA
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pH 8.0, temperature not specified in the publication
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
42350
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1 * 46578 + 1 * 42350, calculated from sequence
43000
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2 * 43000, SDS-PAGE
46578
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1 * 46578 + 1 * 42350, calculated from sequence
90000
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gel filtration
148000
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1:1 protein complex of the heterodimeric dehydratase (89 kDa) and the homodimeric activator (60 kDa), stable only in the presence of AlF4-, gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heterodimer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystals of the dehydratase are grown at 16°C by the vapor diffusion technique from a reservoir solution containing 18-29% (w/v) PEG 3350 and 100 mM BisTris (pH 6.5). The dehydratase is cocrystallized by incubation with 5 mM (R)-2-hydroxyisocaproate 10 min prior to crystallization
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
overexpression in Escherichia coli
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the hadBC and hadI genes from Clostridium difficile are functionally expressed in Escherichia coli. They encode the 2-hydroxyisocaproyl-CoA dehydratase HadBC and its activator HadI
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