Structural Basis for the Activity and Substrate Specificity of Fluoroacetyl-CoA Thioesterase FlK |
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Authors: | Marcio V B Dias Fanglu Huang Dimitri Y Chirgadze Manuela Tosin Dieter Spiteller Emily F V Dry Peter F Leadlay Jonathan B Spencer and Tom L Blundell |
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Institution: | From the ‡Department of Biochemistry and ;§University Chemical Laboratory, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom |
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Abstract: | 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 Thr42, His76, 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 Arg120 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. |
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Keywords: | Acetyl-Coenzyme A Bacterial Metabolism Coenzyme A Crystal Structure Enzyme Mechanisms Protein Structure Site-directed Mutagenesis Fluoroacetyl-Coenzyme A Thioesterase |
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