An isoleucine/leucine residue in the carboxyltransferase domain of acetyl-CoA carboxylase is critical for interaction with aryloxyphenoxypropionate and cyclohexanedione inhibitors

Abstract

cDNA fragments encoding the carboxyltransferase domain of the multidomain plastid acetyl-CoA carboxylase (ACCase) from herbicide-resistant maize and from herbicide-sensitive and herbicide-resistant Lolium rigidum were cloned and sequenced. A Leu residue was found in ACCases from herbicide-resistant plants at a position occupied by Ile in all ACCases from sensitive grasses studied so far. Leu is present at the equivalent position in herbicide-resistant ACCases from other eukaryotes. Chimeric ACCases containing a 1000-aa fragment of two ACCase isozymes found in a herbicide-resistant maize were expressed in a yeast ACC1 null mutant to test herbicide sensitivity of the enzyme in vivo and in vitro. One of the enzymes was resistant/tolerant, and one was sensitive to haloxyfop and sethoxydim, rendering the gene-replacement yeast strains resistant and sensitive to these compounds, respectively. The sensitive enzyme has an Ile residue, and the resistant one has a Leu residue at the putative herbicide-binding site. Additionally, a single Ile to Leu replacement at an equivalent position changes the wheat plastid ACCase from sensitive to resistant. The effect of the opposite substitution, Leu to Ile, makes Toxoplasma gondii apicoplast ACCase resistant to haloxyfop and clodinafop. In this case, inhibition of the carboxyltransferase activity of ACCase (second half-reaction) of a large fragment of the Toxoplasma enzyme expressed in Escherichia coli was tested. The critical amino acid residue is located close to a highly conserved motif of the carboxyltransferase domain, which is probably a part of the enzyme active site, providing the basis for the activity of fop and dim herbicides.

Figure 1

Structure of chimeric ACCases expressed in yeast. Construct names reflect composition of the encoded proteins: C, wheat cytosolic ACCase; P, wheat plastid ACCase; M, maize plastid ACCase. Numbers indicate approximate contribution (%) of each segment. W-t, wild-type (Ile); mut, mutant (Leu). The origin of ACCase coding sequences and yeast DNA segments is indicated by different patterns. The major functional domains (biotin carboxylase and carboxyltransferase) and the biotin attachment sites are located as described before (–19). The herbicide sensitivity domain was identified before (9). The approximate position of the T. gondii ACCase fragment (amino acid residues 1,861–2,609 of ACC1, GenBank accession no. AF157612) expressed in E. coli, relative to wheat ACCase is indicated with a horizontal arrow. Locations of key restriction sites used in the constructions are shown. Alignment of amino acid sequences of multidomain ACCases at the site of the critical Ile/Leu residue (in bold) and an approximate location of this domain in ACCase are shown. The fragment shown corresponds to residues 1,752–1,864 of wheat plastid ACCase ORF (AF029895). Names of plastid ACCases of grasses are underlined. Dots indicate residues identical to those in Triticum aestivum (AF029895). Dashes indicate gaps.

Figure 2

Growth inhibition of yeast gene-replacement strains expressing chimeric ACCases by sethoxydim (A) and haloxyfop (B). The structures of chimeral genes are shown in Fig. 1, and growth properties of the yeast strains are shown in Table 1. The strains are identified by the source of their herbicide-binding ACCase domain. Average values for strains containing the same construct (Table 1), each an average of three measurements for each strain, are shown with error bars. Growth without herbicide is set at 1.00. Inhibition of chimeric ACCases by sethoxydim (C) and haloxyfop (D). Protein extracts were prepared from the yeast gene-replacement haploid strains. One strain of each type was tested for resistance to herbicides (C₃₀M₅₀P₂₀ MR2 strain a and C₃₀M₅₀P₂₀ MR3 strain a, Table 1). Chimeric ACCases are identified by the source of their herbicide-binding domain. Average results of two independent experiments are shown with error bars. Activity without herbicide is set at 1.00.

Figure 3

Effect of a single amino acid substitution in T. gondii ACCase on interaction with aryloxyphenoxypropionate and cyclohexanedione inhibitors. Reverse carboxyltransferase activity (ACCase second half-reaction) was measured by using protein fragments expressed in E. coli. Protein structure (ACC1/ct2) and the mutation site are shown in Fig. 1. Average results of two independent experiments, each with replicates, are shown with error bars. Activity without herbicide is set at 1.00. W-t, wild-type (Leu); mut, mutant (Ile).