Molecular bases for sensitivity to acetyl-coenzyme A carboxylase inhibitors in black-grass

Abstract

In grasses, residues homologous to residues Ile-1,781 and Ile-2,041 in the carboxyl-transferase (CT) domain of the chloroplastic acetyl-coenzyme A (CoA) carboxylase (ACCase) from the grass weed black-grass (Alopecurus myosuroides [Huds.]) are critical determinants for sensitivity to two classes of ACCase inhibitors, aryloxyphenoxypropionates (APPs) and cyclohexanediones. Using natural mutants of black-grass, we demonstrated through a molecular, biological, and biochemical approach that residues Trp-2,027, Asp-2,078, and Gly-2,096 are also involved in sensitivity to ACCase inhibitors. In addition, residues Trp-2,027 and Asp-2,078 are very likely involved in CT activity. Using three-dimensional modeling, we found that the side chains of the five residues are adjacent, located at the surface of the inside of the cavity of the CT active site, in the vicinity of the binding site for APPs. Residues 1,781 and 2,078 are involved in sensitivity to both APPs and cyclohexanediones, whereas residues 2,027, 2,041, and 2,096 are involved in sensitivity to APPs only. This suggests that the binding sites for these two classes of compounds are overlapping, although distinct. Comparison of three-dimensional models for black-grass wild-type and mutant CTs and for CTs from organisms with contrasted sensitivity to ACCase inhibitors suggested that inhibitors fitting into the cavity of the CT active site of the chloroplastic ACCase from grasses to reach their active sites may be tight. The three-dimensional shape of this cavity is thus likely of high importance for the efficacy of ACCase inhibitors.

Figure 1.

Inhibition of ACCase activity of wild-type (population 00-017; ♦), Cys-2,027 (population Cys-2,027-F1; ▴), Gly-2,078 (population Gly-2,078-F1; •), and Ala-2,096 (population Ala-2,096-F1; ▪) ACCase alleles by clethodim and cycloxydim (CHDs) and by fenoxaprop, diclofop, clodinafop, and haloxyfop (APPs). Averages of two independent experiments are shown with error bars. ACCase activity is expressed as a percentage of ACCase activity without inhibitor for each allele.

Figure 2.

Ribbon diagram views of the inside of the CT active-site cavity in a three-dimensional model for black-grass chloroplastic ACCase. Side-chain colors: Green, APP-binding site; magenta, variable amino acid, residue present in wild-type black-grass ACCase; blue, variable amino acid, residue present in mutant black-grass ACCase. Hydrogen atoms and side chains not discussed in the text are not shown for clarity. A, Views of the inside of the active-site cavity part formed by monomer C in Ile-1,781 and Leu-1,781 ACCase alleles. Monomer B is not shown. B, Views of the inside of the active-site cavity part formed by monomer B in wild-type ACCase allele after a 180° rotation with respect to the views of monomer C. Monomer C is not shown.

Figure 3.

Ribbon diagram views of the CT active-site cavity on monomer B in three-dimensional models for black-grass chloroplastic, C. cryptica, alfalfa cytosolic, and rat (D) ACCases. Side-chain colors: Green, APP-binding site and variable amino acid identified in black-grass ACCase; magenta, amino acid variable between organisms; blue, amino acid variable between organisms, residue differing from that present in black-grass at this position (see Table III). Hydrogen atoms and side chains not discussed in the text are not shown for clarity. Monomer C is not shown.