title: Structural basis for the activity and substrate specificity of fluoroacetyl-CoA thioesterase FlK.
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 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.
authors.affiliation: Department of Biochemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom.
authors.forename: Marcio V B
authors.initials: MV
authors.lastname: Dias
authors.forename: Fanglu
authors.initials: F
authors.lastname: Huang
authors.forename: Dimitri Y
authors.initials: DY
authors.lastname: Chirgadze
authors.forename: Manuela
authors.initials: M
authors.lastname: Tosin
authors.forename: Dieter
authors.initials: D
authors.lastname: Spiteller
authors.forename: Emily F V
authors.initials: EF
authors.lastname: Dry
authors.forename: Peter F
authors.initials: PF
authors.lastname: Leadlay
authors.forename: Jonathan B
authors.initials: JB
authors.lastname: Spencer
authors.forename: Tom L
authors.initials: TL
authors.lastname: Blundell
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism*; D055162:Biocatalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity; D013869:Thiolester Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D013912:Threonine / Q000235:genetics
chemical_list: D050505:Mutant Proteins; D013912:Threonine; C031696:fluoroacetyl-coenzyme A; D000105:Acetyl Coenzyme A; D013869:Thiolester Hydrolases