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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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Active sites of ligand-bound FlK with 2Fo − 2Fc electron density map for T42SFlK·AcCoA (A), WtFlK·FAcCPan (B), and WtFlK·FAcOPan (C). The molecules of AcCoA, FAcCPan, and FAcOPan are all shown in a deep purple color. Green and orange colors indicate residues from the two protomers of the dimer.
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Figure 3: Active sites of ligand-bound FlK with 2Fo − 2Fc electron density map for T42SFlK·AcCoA (A), WtFlK·FAcCPan (B), and WtFlK·FAcOPan (C). The molecules of AcCoA, FAcCPan, and FAcOPan are all shown in a deep purple color. Green and orange colors indicate residues from the two protomers of the dimer.

Mentions: Although attempts to co-crystallize FlK with FAcCoA, CoA, or pantothenic acid failed, we successfully obtained crystals of T42SFlK with bound AcCoA and WtFlK in complex with two analogues of fluoroacetyl-pantetheine, FAcCPan, and FAcOPan. The sulfur atom of the thioester is replaced by a methylene in FAcCPan and by an oxygen atom in FAcOPan (Fig. 3). Comparison of the electron density maps of the crystals obtained from co-crystallization of WtFlK with FAc and CoA indicates that only FAc was bound. Comparisons of the structure of WtFlK without bound ligand with those of WtFlK·FAcCPan, WtFlK·FAcOPan, and T42SFlK·AcCoA have provided interesting insights into the structural basis of substrate recognition by FlK.


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)

Active sites of ligand-bound FlK with 2Fo − 2Fc electron density map for T42SFlK·AcCoA (A), WtFlK·FAcCPan (B), and WtFlK·FAcOPan (C). The molecules of AcCoA, FAcCPan, and FAcOPan are all shown in a deep purple color. Green and orange colors indicate residues from the two protomers of the dimer.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2903362&req=5

Figure 3: Active sites of ligand-bound FlK with 2Fo − 2Fc electron density map for T42SFlK·AcCoA (A), WtFlK·FAcCPan (B), and WtFlK·FAcOPan (C). The molecules of AcCoA, FAcCPan, and FAcOPan are all shown in a deep purple color. Green and orange colors indicate residues from the two protomers of the dimer.
Mentions: Although attempts to co-crystallize FlK with FAcCoA, CoA, or pantothenic acid failed, we successfully obtained crystals of T42SFlK with bound AcCoA and WtFlK in complex with two analogues of fluoroacetyl-pantetheine, FAcCPan, and FAcOPan. The sulfur atom of the thioester is replaced by a methylene in FAcCPan and by an oxygen atom in FAcOPan (Fig. 3). Comparison of the electron density maps of the crystals obtained from co-crystallization of WtFlK with FAc and CoA indicates that only FAc was bound. Comparisons of the structure of WtFlK without bound ligand with those of WtFlK·FAcCPan, WtFlK·FAcOPan, and T42SFlK·AcCoA have provided interesting insights into the structural basis of substrate recognition by FlK.

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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