Characterization of (R)-2-Hydroxyisocaproate Dehydrogenase and a Family III Coenzyme A Transferase Involved in Reduction of L-Leucine to Isocaproate by Clostridium difficile

The strictly anaerobic pathogenic bacterium Clostridium difficile occurs in the human gut and is able to thrive from fermentation of leucine. Thereby the amino acid is both oxidized to isovalerate plus CO₂ and reduced to isocaproate. In the reductive branch of this pathway, the dehydration of (R)-2-...

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Bibliographic Details
Published in:Applied and Environmental Microbiology 2006-09, Vol.72 (9), p.6062-6069
Main Authors: Kim, Jihoe, Darley, Daniel, Selmer, Thorsten, Buckel, Wolfgang
Format: Article
Language:English
Subjects:
Alcohol Oxidoreductases - chemistry
Alcohol Oxidoreductases - genetics
Alcohol Oxidoreductases - metabolism
Amino Acid Sequence
Amino acids
Bacteria
Base Sequence
Biological and medical sciences
Caproates - metabolism
Catalytic Domain - genetics
Cloning, Molecular
Clostridium difficile
Clostridium difficile - enzymology
Clostridium difficile - genetics
Clostridium difficile - metabolism
Clostridium difficile - pathogenicity
Coenzyme A-Transferases - antagonists & inhibitors
Coenzyme A-Transferases - classification
Coenzyme A-Transferases - genetics
Coenzyme A-Transferases - metabolism
Dehydration
DNA, Bacterial - genetics
Enzymology and Protein Engineering
Escherichia coli
Fermentation
Fundamental and applied biological sciences. Psychology
Genes, Bacterial
Humans
Kinetics
Leucine - metabolism
Microbiology
Molecular Sequence Data
Mutagenesis, Site-Directed
Oxidation-Reduction
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Sequence Homology, Amino Acid
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Substrate Specificity
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Summary:The strictly anaerobic pathogenic bacterium Clostridium difficile occurs in the human gut and is able to thrive from fermentation of leucine. Thereby the amino acid is both oxidized to isovalerate plus CO₂ and reduced to isocaproate. In the reductive branch of this pathway, the dehydration of (R)-2-hydroxyisocaproyl-coenzyme A (CoA) to (E)-2-isocaprenoyl-CoA is probably catalyzed via radical intermediates. The dehydratase requires activation by an ATP-dependent one-electron transfer (J. Kim, D. Darley, and W. Buckel, FEBS J. 272:550-561, 2005). Prior to the dehydration, a dehydrogenase and a CoA transferase are supposed to be involved in the formation of (R)-2-hydroxyisocaproyl-CoA. Deduced amino acid sequences of ldhA and hadA from the genome of C. difficile showed high identities to D-lactate dehydrogenase and family III CoA transferase, respectively. Both putative genes encoding the dehydrogenase and CoA transferase were cloned and overexpressed in Escherichia coli; the recombinant Strep tag II fusion proteins were purified to homogeneity and characterized. The substrate specificity of the monomeric LdhA (36.5 kDa) indicated that 2-oxoisocaproate (Km = 68 μM, kcat = 31 s⁻¹) and NADH were the native substrates. For the reverse reaction, the enzyme accepted (R)- but not (S)-2-hydroxyisocaproate and therefore was named (R)-2-hydroxyisocaproate dehydrogenase. HadA showed CoA transferase activity with (R)-2-hydroxyisocaproyl-CoA as a donor and isocaproate or (E)-2-isocaprenoate as an acceptor. By site-directed mutagenesis, the conserved D171 was identified as an essential catalytic residue probably involved in the formation of a mixed anhydride with the acyl group of the thioester substrate. However, neither hydroxylamine nor sodium borohydride, both of which are inactivators of the CoA transferase, modified this residue. The dehydrogenase and the CoA transferase fit well into the proposed pathway of leucine reduction to isocaproate.
ISSN:0099-2240
1098-5336
DOI:10.1128/AEM.00772-06