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Information on EC 4.2.1.157 - (R)-2-hydroxyisocaproyl-CoA dehydratase and Organism(s) Clostridioides difficile and UniProt Accession Q5U924
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4.2.1.157 (R)-2-hydroxyisocaproyl-CoA dehydrataseIUBMB Comments
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.
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Word Map
- 4.2.1.157
-
mycolic
-
hadab
- tuberculosis
- fas-ii
-
ketyl
- difficile
- clostridia
-
2-hydroxyacyl-coa
- thiacetazone
-
antitubercular
- isoxyl
- nitrogenase
-
3-hydroxyacyl
-
2-hydroxyglutaryl-coa
- benzoyl-coa
-
oxygen-sensitive
- fermentans
-
acidaminococcus
- symbiosum
The taxonomic range for the selected organisms is: Clostridioides difficile
The expected taxonomic range for this enzyme is: Bacteria, Archaea
The expected taxonomic range for this enzyme is: Bacteria, Archaea
Synonyms
hadbc, (r)-2-hydroxyisocaproyl-coa dehydratase, 2-hydroxyisocaproyl-coa dehydratase, hadbc dehydratase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
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.
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(R)-2-hydroxy-4-methylpentanoyl-CoA
4-methylpent-2-enoyl-CoA + H2O
i.e. (R)-2-hydroxyisocaproyl-CoA. The enzyme is involved in the reductive branch of L-leucine fermentation
i.e. isocaprenoyl-CoA
-
?
(R)-2-hydroxy-4-methylpentanoyl-CoA
4-methylpent-2-enoyl-CoA + H2O
the enzyme is involved in fermentation of L-leucine
-
-
?
(R)-2-hydroxy-4-methylpentanoyl-CoA
4-methylpent-2-enoyl-CoA + H2O
i.e. (R)-2-hydroxyisocaproyl-CoA. The enzyme is specific for the R-isomer. The reduced activator transfers one electron to the dehydratase concomitant with hydrolysis of ATP. The activated dehydratase is separated from the activator and ATP. It catalyzes about 10000 dehydration turnovers until the enzyme becomes inactive. Adding activator, ATP, MgCl2, dithionite and dithioerythritol reactivates the enzyme. The enzymatic elimination of H2O probably occurs in syn-fashion. In assays using (E)-isocaprenoyl-CoA as substrate, no activity can be observed suggesting that the dehydration is irreversible under these conditions or the Z-isomer is the correct product
i.e. isocaprenoyl-CoA
-
?
(R)-2-hydroxy-4-methylpentanoyl-CoA
4-methylpent-2-enoyl-CoA + H2O
the catalyzed reaction, an atypical alpha/beta-dehydration, depends on the reductive formation of ketyl radicals on the substrate generated by injection of a single electron from the ATP-dependent activator protein
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(R)-2-hydroxy-4-methylpentanoyl-CoA
4-methylpent-2-enoyl-CoA + H2O
i.e. (R)-2-hydroxyisocaproyl-CoA. The enzyme is involved in the reductive branch of L-leucine fermentation
i.e. isocaprenoyl-CoA
-
?
(R)-2-hydroxy-4-methylpentanoyl-CoA
4-methylpent-2-enoyl-CoA + H2O
the enzyme is involved in fermentation of L-leucine
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
[4Fe-4S]-center
-
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
LITERATURE
Iron
Zn2+
besides varying amounts of zinc, other metal ions, particularly molybdenum, are not detected in the dehydratase
[4Fe-4S] center
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
Iron
the enzyme contains 5.7 nonheme iron and 6.1 acid-labile sulfur, determined by chemical analysis
Iron
-
the complex of the heterodimeric dehydratase and the homodimeric activator complex contains per mol 8.5 mol Fe corresponding to the sum of 5.0 mol Fe/mol dehydratase and 3.7 mol Fe/mol activator
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
AlF4-
-
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
-
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
LITERATURE
IMAGE
additional information
-
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
-
activator HadI
the activator of the 2-hydroxyisocaproyl-CoA dehydratase from Clostridium difficile is a homodimeric [4Fe-4S] cluster containing ATPase which is bound in the dimer interface. The crystal structures of the Mg-ADP, Mg-ADPNP, and nucleotide-free states of the reduced activator have been solved at 1.6-3.0 A resolution. Abstraction of the nonacidic beta-proton of the 2-hydroxyacyl-CoA compounds is achieved by the reductive generation of ketyl radicals on the substrate, which is initiated by the transfer of an electron at low redox potentials. The highly energetic electron needed on the dehydratase is donated by a [4Fe-4S] cluster containing ATPase, termed activator. ATP-hydrolysis may not be necessary for electron transfer between the activator and dehydratase. The different propensities of the ATP- versus ADP-bound activator to form a complex with the dehydratase would be in agreement with electron transfer already being enabled by binding of the ATP-bound activator to the dehydratase. ATP-hydrolysis would then be needed to regenerate the ADP-bound state of the activator whose low affinity for the dehydratase would trigger complex dissociation allowing the cycle of reduction of the activator and ATP-induced complex formation between activator and dehydratase to start again
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activator HadI
the homodimeric activator is extremely oxygen-sensitive. Recombinant HadI activates the dehydratase in the presence of ATP, MgCl2 and a one-electron reducing agent titanium(III) citrate or dithionite
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.05 - 0.08
(R)-2-hydroxy-4-methylpentanoyl-CoA
pH 8.0, temperature not specified in the publication
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
hadI, activator of 2-hydroxyisocaproyl-CoA dehydratase
UniProt
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
metabolism
metabolism
the enzyme is involved in L-leucine fermentation
metabolism
the enzyme is involved in the reductive branch of L-leucine fermentation
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
148000
-
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
42350
1 * 46578 + 1 * 42350, calculated from sequence
46578
1 * 46578 + 1 * 42350, calculated from sequence
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
heterodimer
heterodimer
1 * 46578 + 1 * 42350, calculated from sequence
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
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Knauer, S.H.; Buckel, W.; Dobbek, H.
On the ATP-dependent activation of the radical enzyme (R)-2-hydroxyisocaproyl-CoA dehydratase
Biochemistry
51
6609-6622
2012
Clostridioides difficile (Q5U925), Clostridioides difficile
Kim, J.; Darley, D.; Buckel, W.
2-Hydroxyisocaproyl-CoA dehydratase and its activator from Clostridium difficile
FEBS J.
272
550-561
2005
Clostridioides difficile (Q5U923 and Q5U924), Clostridioides difficile
Knauer, S.H.; Buckel, W.; Dobbek, H.
Structural basis for reductive radical formation and electron recycling in (R)-2-hydroxyisocaproyl-CoA dehydratase
J. Am. Chem. Soc.
133
4342-4347
2011
Clostridioides difficile (Q5U923 and Q5U924), Clostridioides difficile
Kim, J.; Lu, Y.; Buckel, W.
ATP- and redox-induced conformational changes in the activator of the radical enzyme 2-hydroxyisocaproyl-CoA dehydratase
C. R. Chimie
10
742-747
2007
Clostridioides difficile
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