Xenobiotic-hydrolysing esterases from crops and Arabidopses: characterisation of a S-formylglutathione hydrolase

Abstract

Esterases represent an ancient family of enzymes, found across all kingdoms, which have diverged and occupied a wide range of functional niches. Because of their biochemical diversity and electrophoretic variability, esterases are widely used as genetic markers in gel electrophoresis assays, yet they remain ill-defined. The aim of this study is to characterise esterases from plants with roles in both normal cellular metabolism as well as in xenobiotic metabolism. A survey of esterase activities in four species of crop plants (Triticum aestivum, Zea mays, Glycine max and Oryza sativa) and Arabidopsis thaliana has shown that most of the esterases were typical carboxylesterases containing a catalytically active serine residue. Esterases varied considerably between plants according to their electrophoretic mobility and their substrate specificity. However, their sensitivities to inhibitors were broadly similar with serine hydrolase inhibitors, such as paraoxon, abolishing most esterolytic activify. Many pesticides are formulated as hydrophobic esters and once in the plant tissues are subject to ester hydrolysis which can result in either their bioactivation or detoxification. Plants studied showed marked differences in their ability to hydrolyse five herbicides with soybean and maize showing most activity. Moreover, roots of the plants exhibited higher esterase activity towards xenobiotics than the respective shoots. A unique esterase from Arabidopsis thaliana, S-formylglutathione hydrolase (AtSFGH), that is potentially involved in cellular formaldehyde detoxification was cloned and expressed in E. coli. AtSFGR enzyme is a dimmer composed of 31 kDa subunits and has a pI of pH 6. The sequence of AtSFGH shared around 50% homology with SFGHs from other organisms including yeast and man. AtSFGH was tested for its hydrolytic activity toward thioesters of glutathione, namely S-formylglutathione and S-acetylglutathione, as well as carboxylesterase activity toward 4-methylumbelliferyl acetate. Based on inhibitor sensitivity, AtfSFGH was defined as a cysteine-dependant hydrolase due to abolition of its activity by heavy metals and sulphydryl alkylating reagents and its insensitivity to organophosphorous compounds. AtSFGH showed very little activity towards herbicides, the highest being toward 2,4-D methyl (0.14 pkat/mg protein). While sharing a number biochemical characteristics with SFGHs from other organisms, AtSFGH differed from other SFGHs in its ability to hydrolyse 5-acetylglutathione and by not being induced by exposure to chemicals such as phenobarbital.