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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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.
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
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
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
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
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
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
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
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
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
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
Physiological Function of Mycobacterial mtFabD, an Essential Malonyl-CoA:AcpM Transacylase of Type 2 Fatty Acid Synthase FASII, in Yeast mct1Delta Cells.
Mycobacterium tuberculosis strain H37Ra describes a 1 bp insertion in the MRA0648 gene, the orthologue of the Rv0637 (hadC) gene of Mycobacterium tuberculosis strain H37Rv
Mycobacterium tuberculosis strain H37Ra describes a 1 bp insertion in the MRA0648 gene, the orthologue of the Rv0637 (hadC) gene of Mycobacterium tuberculosis strain H37Rv
mutation or deletion of hadC affects the biosynthesis of oxygenated mycolic acids, substantially reducing their production level. Myxolic acid distribution is strongly affected by hadC mutation or deletion in Mtb H37Rv with a decrease in the methoxy-mycolic acid content compensated by an increase in alpha-mycolic acids. Additionally, it causes the loss of atypical extra-long mycolic acids. These events have an impact on the morphotype, cording capacity and biofilm growth of the bacilli as well as on their sensitivity to agents such as rifampicin. Deletion of hadC also leads to a dramatic loss of virulence: an almost 4-log drop of the bacterial load in the lungs and spleens of infected immunodeficient mice. Mycolic acids of Mycobacterium tuberculosis and impact of hadC mutation on their relative distribution, Effect of hadC mutation on the morphotype, biofilm growth, tolerance to stresses and virulence of Mycobacterium tuberculosis, phenotypes, detailed overview
mutation or deletion of hadC does not affect the growth of Mycobacterium tuberculosis. Myxolic acid distribution is strongly affected by hadC mutation or deletion in Mtb H37Ra with a decrease in the methoxy-mycolic acid content compensated by an increase in alpha-mycolic acids. Complementation of Mycobacterium tuberculosis strain H37Ra with either the wild-type hadC (hadCRv) or both wild-type hadB and hadC allele(s) (hadBCRv) from Mycobacterium tuberculosis strain H37Rv results in similar intermediate phenotypes. Effect of hadC mutation on the morphotype, biofilm growth, tolerance to stresses and virulence of Mycobacterium tuberculosis. Phenotypes, detailed overview
mutation or deletion of hadC affects the biosynthesis of oxygenated mycolic acids, substantially reducing their production level. Myxolic acid distribution is strongly affected by hadC mutation or deletion in Mtb H37Rv with a decrease in the methoxy-mycolic acid content compensated by an increase in alpha-mycolic acids. Additionally, it causes the loss of atypical extra-long mycolic acids. These events have an impact on the morphotype, cording capacity and biofilm growth of the bacilli as well as on their sensitivity to agents such as rifampicin. Deletion of hadC also leads to a dramatic loss of virulence: an almost 4-log drop of the bacterial load in the lungs and spleens of infected immunodeficient mice. Mycolic acids of Mycobacterium tuberculosis and impact of hadC mutation on their relative distribution, Effect of hadC mutation on the morphotype, biofilm growth, tolerance to stresses and virulence of Mycobacterium tuberculosis, phenotypes, detailed overview
mutation or deletion of hadC does not affect the growth of Mycobacterium tuberculosis. Myxolic acid distribution is strongly affected by hadC mutation or deletion in Mtb H37Ra with a decrease in the methoxy-mycolic acid content compensated by an increase in alpha-mycolic acids. Complementation of Mycobacterium tuberculosis strain H37Ra with either the wild-type hadC (hadCRv) or both wild-type hadB and hadC allele(s) (hadBCRv) from Mycobacterium tuberculosis strain H37Rv results in similar intermediate phenotypes. Effect of hadC mutation on the morphotype, biofilm growth, tolerance to stresses and virulence of Mycobacterium tuberculosis. Phenotypes, detailed overview
involvement of HadBC in the late elongation steps of mycolic acid biosynthesis, involvement of hadC gene in the biosynthesis of atypical extra-long mycolic acids
involvement of HadBC in the late elongation steps of mycolic acid biosynthesis, involvement of hadC gene in the biosynthesis of atypical extra-long mycolic acids. Gene hadC is not essential for Mycobacterium tuberculosis growth, inactivity of the hadC gene of H37Ra. Mycolic acids are involved in the pathogenicity of mycobacteria suggesting that there might be a link between the mutation in hadC and the attenuation of Mtb H37Ra
involvement of HadBC in the late elongation steps of mycolic acid biosynthesis, involvement of hadC gene in the biosynthesis of atypical extra-long mycolic acids
involvement of HadBC in the late elongation steps of mycolic acid biosynthesis, involvement of hadC gene in the biosynthesis of atypical extra-long mycolic acids. Gene hadC is not essential for Mycobacterium tuberculosis growth, inactivity of the hadC gene of H37Ra. Mycolic acids are involved in the pathogenicity of mycobacteria suggesting that there might be a link between the mutation in hadC and the attenuation of Mtb H37Ra
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
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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
mutation or deletion of hadC, phenotype, overview. The hadC allele of that Mycobacterium tuberculosis strain H37Ra contains the previously reported single nucleotide insertion (C) in a GC rich region at base pair 88 (Rv0637 numbering), that it causes a frameshift in MRA0648, generating an early stop codon. The deletion mutant is termed H37RaDELTAhadC. No complementation is observed after transformation of Mycobacterium tuberculosis H37RvDELTAhadC mutant with hadC allele from Mycobacterium tuberculosis H37Ra (hadCRa), and the complete deletion of this allele in H37Ra does not significantly affect the mycolic distribution. Complementation of Mycobacterium tuberculosis strain H37Ra with either the wild-type hadC (hadCRv) or both wild-type hadB and hadC allele(s) (hadBCRv) from Mycobacterium tuberculosis strain H37Rv results in similar intermediate phenotypes
mutation or deletion of hadC, phenotype, overview. The hadC allele of that Mycobacterium tuberculosis strain H37Ra contains the previously reported single nucleotide insertion (C) in a GC rich region at base pair 88 (Rv0637 numbering), that it causes a frameshift in MRA0648, generating an early stop codon. The deletion mutant is termed H37RaDELTAhadC. No complementation is observed after transformation of Mycobacterium tuberculosis H37RvDELTAhadC mutant with hadC allele from Mycobacterium tuberculosis H37Ra (hadCRa), and the complete deletion of this allele in H37Ra does not significantly affect the mycolic distribution. Complementation of Mycobacterium tuberculosis strain H37Ra with either the wild-type hadC (hadCRv) or both wild-type hadB and hadC allele(s) (hadBCRv) from Mycobacterium tuberculosis strain H37Rv results in similar intermediate phenotypes
mutation or deletion of hadC, phenotype, overview. The hadC allele of that Mycobacterium tuberculosis strain H37Ra contains the previously reported single nucleotide insertion (C) in a GC rich region at base pair 88 (Rv0637 numbering), that it causes a frameshift in MRA0648, generating an early stop codon. The deletion mutant is termed H37RaDELTAhadC. No complementation is observed after transformation of Mycobacterium tuberculosis H37RvDELTAhadC mutant with hadC allele from Mycobacterium tuberculosis H37Ra (hadCRa), and the complete deletion of this allele in H37Ra does not significantly affect the mycolic distribution. Complementation of Mycobacterium tuberculosis strain H37Ra with either the wild-type hadC (hadCRv) or both wild-type hadB and hadC allele(s) (hadBCRv) from Mycobacterium tuberculosis strain H37Rv results in similar intermediate phenotypes
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
A complex of 2-hydroxyisocaproyl-Coenzyme A dehydratase and its activator from clostridium difficile stabilized by aluminium tetrafluoride-adenosine diphosphate