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. 2010 Jul 16;285(29):22495-504.
doi: 10.1074/jbc.M110.107177. Epub 2010 Apr 29.

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

Affiliations

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

Marcio V B Dias et al. J Biol Chem. .

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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Figures

FIGURE 1.
FIGURE 1.
Overall structure of FlK depicted in a schematic representation. A, structure of WtFlK dimer. The two protomers of the dimer are shown in green and orange, respectively. B, tetrameric structure (dimer of dimers) of WtFlK in complex with the substrate analogue FAcCPan. The FAcCPan molecule is shown as sticks. The two protomers of the dimer on the top are indicated in green and orange, respectively. A red/blue color scheme is used for the dimer at the bottom.
FIGURE 2.
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.
FIGURE 3.
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.
FIGURE 4.
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.
FIGURE 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.
FIGURE 6.
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.
FIGURE 7.
FIGURE 7.
Representation of the hydrophobic loop 33FAEFP37 present in the active site of FlK. Shown are the differences in the side chain for residues Phe33, Phe36, and Pro37 for WtFlK·FAc (yellow) and apo-WtFlK (green).

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