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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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Catalytic center of FlK. A, the catalytic triad of FlK is composed of Thr42, His76 (shown as sticks), and a conserved water molecule (Wat) (shown as a sphere). Green and orange colors indicate the two protomers of the dimer, respectively. B, the catalytic triad is located at the dimer interface. The electron density map used was 2Fo − 2Fc.
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Figure 2: Catalytic center of FlK. A, the catalytic triad of FlK is composed of Thr42, His76 (shown as sticks), and a conserved water molecule (Wat) (shown as a sphere). Green and orange colors indicate the two protomers of the dimer, respectively. B, the catalytic triad is located at the dimer interface. The electron density map used was 2Fo − 2Fc.

Mentions: Consistent with the results of the site-directed mutagenesis of FlK described above, the crystal structure of apo-form WtFlK shows that side chains of the candidate catalytic residues Thr42 and His76 in one protomer and that of Glu50 from the adjacent protomer cluster between the dimer interface into the region between the “bun” and the “sausage” (Fig. 2, A and B). As expected, the Oγ1 of Thr42 and the Nδ1 of His76 are within hydrogen bonding distance (2.74 Å). To our surprise, we observed that the carboxylic group of Glu50 is located on the opposite side of Thr42, where it cannot have any direct contact with His76. However, a water molecule (Wat1) is in a perfect position to form a hydrogen bond with the Nδ2 of His76 (2.79 Å). This water molecule is conserved in the crystal structures of wild-type and mutant FlK with or without bound ligands. Although this observation was unexpected, it is in good agreement with our site-directed mutagenesis results. A water molecule replacing the acidic residue in this type of catalytic triad was also found in the HAV-3C gene product, an α-chymotrypsin-like protease produced by the hepatitis A picornavirus (36). The Thr42-His76-Wat1 network is further stabilized by a hydrogen bond between Wat1 and Thr80 side chain hydroxyl and another conserved water molecule (Wat2) that also forms a hydrogen bond with the Phe40-N. Around the His76-Wat1-Thr80/Wat2 network, the side chains from Val39, Phe40, Ala78, Ala79, and Ile113 form a hydrophobic shield, protecting the network from attack by solvent molecules. Based on our structural evidence, we now propose that the catalytic triad in FlK comprises Thr42-His76-water (Fig. 2A), located at the interface between two protomers with two active sites in each FlK dimer (Fig. 2B).


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)

Catalytic center of FlK. A, the catalytic triad of FlK is composed of Thr42, His76 (shown as sticks), and a conserved water molecule (Wat) (shown as a sphere). Green and orange colors indicate the two protomers of the dimer, respectively. B, the catalytic triad is located at the dimer interface. The electron density map used was 2Fo − 2Fc.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Catalytic center of FlK. A, the catalytic triad of FlK is composed of Thr42, His76 (shown as sticks), and a conserved water molecule (Wat) (shown as a sphere). Green and orange colors indicate the two protomers of the dimer, respectively. B, the catalytic triad is located at the dimer interface. The electron density map used was 2Fo − 2Fc.
Mentions: Consistent with the results of the site-directed mutagenesis of FlK described above, the crystal structure of apo-form WtFlK shows that side chains of the candidate catalytic residues Thr42 and His76 in one protomer and that of Glu50 from the adjacent protomer cluster between the dimer interface into the region between the “bun” and the “sausage” (Fig. 2, A and B). As expected, the Oγ1 of Thr42 and the Nδ1 of His76 are within hydrogen bonding distance (2.74 Å). To our surprise, we observed that the carboxylic group of Glu50 is located on the opposite side of Thr42, where it cannot have any direct contact with His76. However, a water molecule (Wat1) is in a perfect position to form a hydrogen bond with the Nδ2 of His76 (2.79 Å). This water molecule is conserved in the crystal structures of wild-type and mutant FlK with or without bound ligands. Although this observation was unexpected, it is in good agreement with our site-directed mutagenesis results. A water molecule replacing the acidic residue in this type of catalytic triad was also found in the HAV-3C gene product, an α-chymotrypsin-like protease produced by the hepatitis A picornavirus (36). The Thr42-His76-Wat1 network is further stabilized by a hydrogen bond between Wat1 and Thr80 side chain hydroxyl and another conserved water molecule (Wat2) that also forms a hydrogen bond with the Phe40-N. Around the His76-Wat1-Thr80/Wat2 network, the side chains from Val39, Phe40, Ala78, Ala79, and Ile113 form a hydrophobic shield, protecting the network from attack by solvent molecules. Based on our structural evidence, we now propose that the catalytic triad in FlK comprises Thr42-His76-water (Fig. 2A), located at the interface between two protomers with two active sites in each FlK dimer (Fig. 2B).

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