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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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Overall structure of T42SFlK in complex with AcCoA. The two protomers of the T42SFlK dimer are represented in green and orange colors, respectively. A, 2Fo − 2Fc electron density map shows that AcCoA is bound between two protomers of the active FlK dimer. B, AcCoA is sandwiched between the long β-sheet and two small α-helices formed by residues 17–42. The long β-sheet and two small α-helices of protomer A and B are shown in light blue and light purple, respectively. C, representation of the molecular surface shows that only the acetyl and β-mercaptoethylamine moieties from AcCoA are buried in the active site of the protein.
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Figure 4: Overall structure of T42SFlK in complex with AcCoA. The two protomers of the T42SFlK dimer are represented in green and orange colors, respectively. A, 2Fo − 2Fc electron density map shows that AcCoA is bound between two protomers of the active FlK dimer. B, AcCoA is sandwiched between the long β-sheet and two small α-helices formed by residues 17–42. The long β-sheet and two small α-helices of protomer A and B are shown in light blue and light purple, respectively. C, representation of the molecular surface shows that only the acetyl and β-mercaptoethylamine moieties from AcCoA are buried in the active site of the protein.

Mentions: In the T42SFlK·AcCoA complex, the AcCoA is sandwiched between the long β-sheet and residues 17–42 that form two small α-helices (Fig. 4B). However, only the acetyl and β-mercaptoethylamine moieties from acetyl-CoA are buried in the protein, whereas the pantothenic acid and the 3′-phosphoryl-ADP are exposed to solvent, allowing considerable flexibility (Fig. 4, A and B). Three of the four protomers present in the asymmetric unit have a bound AcCoA, and in one of them, it was possible to fit the complete molecule, despite the poor electron density for the 3′-phosphoryl-ADP moiety, possibly due to the high flexibility in the solvent. The overall structure of FlK does not change significantly as a result of the T42S point mutation and the binding of AcCoA, but the AcCoA has forced Wat4 and Wat5 out of the active site cavity. The acetyl methyl group of the AcCoA occupies the position of Wat5, and the thioester carbonyl oxygen interacts via a hydrogen bond to Ser42-N. The orientations of the three bound AcCoA molecules at the active site are significantly different, reflecting a dynamic AcCoA-FlK interaction process and the flexibility of the substrate binding site. The acetyl carbonyl carbons of the three bound AcCoA molecules are all in the wrong orientation with respect to Oγ of Ser42 to allow nucleophilic attack. This explains the inability of FlK to hydrolyze AcCoA and implies that the presence of the fluorine in FAcCoA is crucial for substrate recognition.


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)

Overall structure of T42SFlK in complex with AcCoA. The two protomers of the T42SFlK dimer are represented in green and orange colors, respectively. A, 2Fo − 2Fc electron density map shows that AcCoA is bound between two protomers of the active FlK dimer. B, AcCoA is sandwiched between the long β-sheet and two small α-helices formed by residues 17–42. The long β-sheet and two small α-helices of protomer A and B are shown in light blue and light purple, respectively. C, representation of the molecular surface shows that only the acetyl and β-mercaptoethylamine moieties from AcCoA are buried in the active site of the protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Overall structure of T42SFlK in complex with AcCoA. The two protomers of the T42SFlK dimer are represented in green and orange colors, respectively. A, 2Fo − 2Fc electron density map shows that AcCoA is bound between two protomers of the active FlK dimer. B, AcCoA is sandwiched between the long β-sheet and two small α-helices formed by residues 17–42. The long β-sheet and two small α-helices of protomer A and B are shown in light blue and light purple, respectively. C, representation of the molecular surface shows that only the acetyl and β-mercaptoethylamine moieties from AcCoA are buried in the active site of the protein.
Mentions: In the T42SFlK·AcCoA complex, the AcCoA is sandwiched between the long β-sheet and residues 17–42 that form two small α-helices (Fig. 4B). However, only the acetyl and β-mercaptoethylamine moieties from acetyl-CoA are buried in the protein, whereas the pantothenic acid and the 3′-phosphoryl-ADP are exposed to solvent, allowing considerable flexibility (Fig. 4, A and B). Three of the four protomers present in the asymmetric unit have a bound AcCoA, and in one of them, it was possible to fit the complete molecule, despite the poor electron density for the 3′-phosphoryl-ADP moiety, possibly due to the high flexibility in the solvent. The overall structure of FlK does not change significantly as a result of the T42S point mutation and the binding of AcCoA, but the AcCoA has forced Wat4 and Wat5 out of the active site cavity. The acetyl methyl group of the AcCoA occupies the position of Wat5, and the thioester carbonyl oxygen interacts via a hydrogen bond to Ser42-N. The orientations of the three bound AcCoA molecules at the active site are significantly different, reflecting a dynamic AcCoA-FlK interaction process and the flexibility of the substrate binding site. The acetyl carbonyl carbons of the three bound AcCoA molecules are all in the wrong orientation with respect to Oγ of Ser42 to allow nucleophilic attack. This explains the inability of FlK to hydrolyze AcCoA and implies that the presence of the fluorine in FAcCoA is crucial for substrate recognition.

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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