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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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Proposed FAcCoA coordination at the active site of FlK. A, a stick model with FAcCoA shown in silver and residues from the two protomers represented in yellow and green colors, respectively. B, a scheme with FAcCoA (red) and active site residues (black). Hydrogen bonding interactions are denoted by dotted lines with distance in Å.
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Figure 5: Proposed FAcCoA coordination at the active site of FlK. A, a stick model with FAcCoA shown in silver and residues from the two protomers represented in yellow and green colors, respectively. B, a scheme with FAcCoA (red) and active site residues (black). Hydrogen bonding interactions are denoted by dotted lines with distance in Å.

Mentions: We have modeled a FAcCoA molecule into WtFlK (Fig. 5), based on the crystal structures of FlK in complex with various ligands, especially with AcCoA. The minimum energy was calculated using the SYBYL 8.1.1.09097 program. In a similar way to the AcCoA bound in T42SFlK, the 3′-phosphoryl-ADP region of the docked FAcCoA is at the enzyme surface in front of the entrance of the tunnel leading to the active site. The rest of the molecule extends into the tunnel, where the pantetheinyl moiety interacts through its N12, O9, N14, and C19, respectively, with the main chain oxygen and the side chain of His76, Ala79-N, and Phe128-Cϵ1. The hydrogen bond between the side chains of Arg120 and Glu50 is broken due to the flip of the carboxyl group of the Glu50. The fluorine is placed at a hydrogen bonding distance from Gly69-N and the guanidinium group of Arg120. Mutation of the Arg120 to Ala120 yielded a completely insoluble protein (data not shown), consistent with the involvement of Arg120 in maintaining the conformation of FlK. Thr42-Oγ is now located closer (3.79 Å) to the thioester carbonyl carbon and at a better, although not optimal, position for a possible nucleophilic attack. The Thr42-N that is close (2.81 Å) to the thioester carbonyl oxygen could act as an oxyanion hole for stabilization of the tetrahedral acyl-enzyme intermediate (Fig. 5).


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)

Proposed FAcCoA coordination at the active site of FlK. A, a stick model with FAcCoA shown in silver and residues from the two protomers represented in yellow and green colors, respectively. B, a scheme with FAcCoA (red) and active site residues (black). Hydrogen bonding interactions are denoted by dotted lines with distance in Å.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Proposed FAcCoA coordination at the active site of FlK. A, a stick model with FAcCoA shown in silver and residues from the two protomers represented in yellow and green colors, respectively. B, a scheme with FAcCoA (red) and active site residues (black). Hydrogen bonding interactions are denoted by dotted lines with distance in Å.
Mentions: We have modeled a FAcCoA molecule into WtFlK (Fig. 5), based on the crystal structures of FlK in complex with various ligands, especially with AcCoA. The minimum energy was calculated using the SYBYL 8.1.1.09097 program. In a similar way to the AcCoA bound in T42SFlK, the 3′-phosphoryl-ADP region of the docked FAcCoA is at the enzyme surface in front of the entrance of the tunnel leading to the active site. The rest of the molecule extends into the tunnel, where the pantetheinyl moiety interacts through its N12, O9, N14, and C19, respectively, with the main chain oxygen and the side chain of His76, Ala79-N, and Phe128-Cϵ1. The hydrogen bond between the side chains of Arg120 and Glu50 is broken due to the flip of the carboxyl group of the Glu50. The fluorine is placed at a hydrogen bonding distance from Gly69-N and the guanidinium group of Arg120. Mutation of the Arg120 to Ala120 yielded a completely insoluble protein (data not shown), consistent with the involvement of Arg120 in maintaining the conformation of FlK. Thr42-Oγ is now located closer (3.79 Å) to the thioester carbonyl carbon and at a better, although not optimal, position for a possible nucleophilic attack. The Thr42-N that is close (2.81 Å) to the thioester carbonyl oxygen could act as an oxyanion hole for stabilization of the tetrahedral acyl-enzyme intermediate (Fig. 5).

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