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

Dias MV, Huang F, Chirgadze DY, Tosin M, Spiteller D, Dry EF, Leadlay PF, Spencer JB, Blundell TL - J. Biol. Chem. (2010)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom.

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.

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Structural differences observed in the catalytic site of FlK due to different mutations of Thr42. The active site residues Thr42, His76, and Glu50 of WtFIK are superimposed with their counterpart residues in T42AFIK and T42SFIK. A, Thr/Ala/Ser42 and His76 from protomer I and the Glu50 from protomer II; B, Thr/Ala/Ser42 and His76 from protomer II and the Glu50 from protomer I. Pink, WtFlK; green, T42AFlK; white, T42SFlK. The alternative conformations of His76 in T42A (pale green) and in T42S (yellow) are indicated.
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Figure 6: Structural differences observed in the catalytic site of FlK due to different mutations of Thr42. The active site residues Thr42, His76, and Glu50 of WtFIK are superimposed with their counterpart residues in T42AFIK and T42SFIK. A, Thr/Ala/Ser42 and His76 from protomer I and the Glu50 from protomer II; B, Thr/Ala/Ser42 and His76 from protomer II and the Glu50 from protomer I. Pink, WtFlK; green, T42AFlK; white, T42SFlK. The alternative conformations of His76 in T42A (pale green) and in T42S (yellow) are indicated.

Mentions: The hydrophobic interactions between Thr42-Cγ2 and Ile72-Cγ2 in the adjacent protomer provide a “dry” environment around the catalytic Oγ1 of Thr42, protecting it from attacks by solvent molecules, a feature observed for other enzymes that have similar catalytic triad mechanisms (37). The Thr42-Cγ2 may also restrain the freedom of the substrate in the active site. Loss of this restraint imposed by the Cγ2 in the T42S mutant may allow random misbinding of the substrate, which in turn may result in the substrate inhibition observed in the enzyme assays. In support of this notion, the imidazole group of the His76 in the complex T42AFlK·FAc has a rotational disorder of ∼90° or exists in two alternative conformers not observed in WtFlK structures, and the Ser42 in one of the protomers of the T42SFlK·Ac structure shows a double conformation, demonstrating the increased flexibility of these residues in the absence of the Thr42-Cγ2 and/or -Oγ2 (Fig. 6).


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

Dias MV, Huang F, Chirgadze DY, Tosin M, Spiteller D, Dry EF, Leadlay PF, Spencer JB, Blundell TL - J. Biol. Chem. (2010)

Structural differences observed in the catalytic site of FlK due to different mutations of Thr42. The active site residues Thr42, His76, and Glu50 of WtFIK are superimposed with their counterpart residues in T42AFIK and T42SFIK. A, Thr/Ala/Ser42 and His76 from protomer I and the Glu50 from protomer II; B, Thr/Ala/Ser42 and His76 from protomer II and the Glu50 from protomer I. Pink, WtFlK; green, T42AFlK; white, T42SFlK. The alternative conformations of His76 in T42A (pale green) and in T42S (yellow) are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: Structural differences observed in the catalytic site of FlK due to different mutations of Thr42. The active site residues Thr42, His76, and Glu50 of WtFIK are superimposed with their counterpart residues in T42AFIK and T42SFIK. A, Thr/Ala/Ser42 and His76 from protomer I and the Glu50 from protomer II; B, Thr/Ala/Ser42 and His76 from protomer II and the Glu50 from protomer I. Pink, WtFlK; green, T42AFlK; white, T42SFlK. The alternative conformations of His76 in T42A (pale green) and in T42S (yellow) are indicated.
Mentions: The hydrophobic interactions between Thr42-Cγ2 and Ile72-Cγ2 in the adjacent protomer provide a “dry” environment around the catalytic Oγ1 of Thr42, protecting it from attacks by solvent molecules, a feature observed for other enzymes that have similar catalytic triad mechanisms (37). The Thr42-Cγ2 may also restrain the freedom of the substrate in the active site. Loss of this restraint imposed by the Cγ2 in the T42S mutant may allow random misbinding of the substrate, which in turn may result in the substrate inhibition observed in the enzyme assays. In support of this notion, the imidazole group of the His76 in the complex T42AFlK·FAc has a rotational disorder of ∼90° or exists in two alternative conformers not observed in WtFlK structures, and the Ser42 in one of the protomers of the T42SFlK·Ac structure shows a double conformation, demonstrating the increased flexibility of these residues in the absence of the Thr42-Cγ2 and/or -Oγ2 (Fig. 6).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom.

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.

Show MeSH
Related in: MedlinePlus