Biochemical characterisation of esterases active in hydrolysing xenobiotics in wheat and competing weeds

Esterase activities toward model xenobiotic substrates (p-nitrophenyl acetate, naphthyl acetate) and pesticide esters (diclofop methyl, bromoxynil octanoate, binapacryl) have been compared in crude extracts from wheat (Triticum aestivum L.) and Triticum progenitors of wheat. Esterase activities were also determined in the weeds, wild oat (A vena fatua) and two populations of black-grass (Alopecuvus myosuvoides), one of which (Rothamsted) is susceptible to herbicides, while the other (Peldon) shows cross-resistance to multiple classes of herbicides. Esterase activity toward the model substrates was highest in wheat, while the weeds were more active in hydrolysing the pesticides. Using isoelectric focussing (pH 4-8), 13 proteins with esterase activity toward a-naphthyl acetate could be resolved in hexaploid wheat (genome AABBDD). The pattern of these activities was most similar to that of the diploid progenitor T. tauschii (DD), excepting a major acidic esterase (pI 4.6), which originated from T. uvavtu (AA). Resolved esterase activities in the weeds were distinct from those observed in the Tritcum species. However, unlike the case with other classes of xenobiotic-metabolising enzymes, the complement of esterases in the Peldon and Rothamsted populations of black-grass appeared to be identical. In all species, the more basic esterases ( > pI 5.0) were sensitive to inhibition by organophosphate and carbamate insecticides, suggesting that they were B-class esterases. In contrast, the acidic wheat esterase (pl 4.6) with the greatest activity toward a-naphthyl acetate was insensitive to insecticides. This wheat-specific esterase was purified 7000-fold by a combination of hydrophobic interaction chromatography, gel filtration and anion-exchange chromatography. The purified esterase behaved as a monomeric 45-kDa protein showing high activity toward p-nitrophenyl acetate and x-naphthyl acetate, but limited activity toward the pesticides.