Structural basis for the activity and substrate specificity of fluoroacetyl-CoA thioesterase FlK.

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Dias, Marcio VB 
Huang, Fanglu 
Chirgadze, Dimitri Y 
Tosin, Manuela 
Spiteller, Dieter 

The thioesterase FlK from the fluoroacetate-producing Streptomyces cattleya catalyzes the hydrolysis of fluoroacetyl-coenzyme A. This provides an effective self-defense mechanism, preventing any fluoroacetyl-coenzyme A formed from being further metabolized to 4-hydroxy-trans-aconitate, a lethal inhibitor of the tricarboxylic acid cycle. Remarkably, FlK does not accept acetyl-coenzyme A as a substrate. Crystal structure analysis shows that FlK forms a dimer, in which each subunit adopts a hot dog fold as observed for type II thioesterases. Unlike other type II thioesterases, which invariably utilize either an aspartate or a glutamate as catalytic base, we show by site-directed mutagenesis and crystallography that FlK employs a catalytic triad composed of Thr(42), His(76), and a water molecule, analogous to the Ser/Cys-His-acid triad of type I thioesterases. Structural comparison of FlK complexed with various substrate analogues suggests that the interaction between the fluorine of the substrate and the side chain of Arg(120) located opposite to the catalytic triad is essential for correct coordination of the substrate at the active site and therefore accounts for the substrate specificity.

Acetyl Coenzyme A, Biocatalysis, Catalytic Domain, Crystallography, X-Ray, Hydrophobic and Hydrophilic Interactions, Models, Molecular, Mutagenesis, Site-Directed, Mutant Proteins, Mutation, Protein Structure, Quaternary, Protein Structure, Secondary, Streptomyces, Substrate Specificity, Thiolester Hydrolases, Threonine
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J Biol Chem
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Elsevier BV
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Wellcome Trust (079281/Z/06/Z)