A different mechanism for the inhibition of the carboxyltransferase domain of acetyl-coenzyme A carboxylase by tepraloxydim

Abstract

Acetyl-CoA carboxylases (ACCs) are crucial metabolic enzymes and are attractive targets for drug discovery. Haloxyfop and tepraloxydim belong to two distinct classes of commercial herbicides and kill sensitive plants by inhibiting the carboxyltransferase (CT) activity of ACC. Our earlier structural studies showed that haloxyfop is bound near the active site of the CT domain, at the interface of its dimer, and a large conformational change in the dimer interface is required for haloxyfop binding. We report here the crystal structure at 2.3 A resolution of the CT domain of yeast ACC in complex with tepraloxydim. The compound has a different mechanism of inhibiting the CT activity compared to haloxyfop, as well as the mammalian ACC inhibitor CP-640186. Tepraloxydim probes a different region of the dimer interface and requires only small but important conformational changes in the enzyme, in contrast to haloxyfop. The binding mode of tepraloxydim explains the structure-activity relationship of these inhibitors, and provides a molecular basis for their distinct sensitivity to some of the resistance mutations, as compared to haloxyfop. Despite the chemical diversity between haloxyfop and tepraloxydim, the compounds do share two binding interactions to the enzyme, which may be important anchoring points for the development of ACC inhibitors.

Fig. 1.

Crystal structure of CT domain in complex with tepraloxydim. (A) Chemical structures of the herbicides haloxyfop and tepraloxydim. (B) Schematic drawing of the structure of yeast CT domain dimer in complex with tepraloxydim. The N domains of the two monomers are colored in cyan and magenta, while the C domains are colored in yellow and green. The inhibitor is shown in stick models, in dark brown for carbon atoms. The CoA molecule is shown for reference in gray (16). The structure figures were produced with Ribbons (34), Grasp (35), and PyMOL (www.pymol.org).

Fig. 2.

The binding mode of tepraloxydim. (A) Final omit F_o–F_c electron density at 2.3-Å resolution for tepraloxydim, contoured at 3σ. (B) Stereographic drawing showing the binding site for tepraloxydim. The N domain of one monomer is colored in cyan, and the C domain of the other monomer in green. The side chains of residues in the binding site are shown in yellow and magenta, respectively. The side chain of Leu-1705 is shown in red. Hydrogen bonds from the inhibitor to the protein or waters are indicated with thin red lines. Several other water molecules in the binding site are also shown as red spheres. (C) Schematic drawing of the interactions between tepraloxydim and the CT domain.

Fig. 3.

Conformational changes in the CT domain upon inhibitor binding. (A) Structural overlay of the CT domain free enzyme (in gray) and the tepraloxydim complex (in cyan and green for the N and C domains) near the inhibitor binding site. Side chains in the binding site with large conformational changes are also shown. Phe-1956′ is shown for reference. The position of CoA is also shown (in gray). The shift in the position of the α5′ helix is indicated with the red arrow. (B) Molecular surface of the binding site in the tepraloxydim complex, colored in cyan for the N domain and green for the C domain. (C) Molecular surface of the active site of the free enzyme. The model of tepraloxydim is included for reference, and the O-substituent of the oxime moiety is in steric clash with the enzyme. For panels B and C, residues 1759–1772 and 2026′–2098′ have been removed to give a better view of the binding site.

Fig. 4.

Tepraloxydim has a different mechanism of inhibiting the CT domain. Overlay of the binding modes of tepraloxydim (in dark brown) and haloxyfop (in black). The distances between the two oxygen atoms and two methyl groups in the inhibitors are indicated.