Structure activity studies with xenobiotic substrates using carboxylesterases isolated from Arabidopsis thaliana Ian Cummins, Marie Landrum, Patrick G. Steel, Robert Edwards * Centre for Bioactive Chemistry, Department of Chemistry, Durham University, Durham DH1 3LE, United Kingdom Received 20 October 2006; received in revised form 13 December 2006 Available online 31 January 2007 Abstract Carboxylesterases (CXEs) catalyse the hydrolysis of xenobiotics and natural products radically altering their biological activities. Whereas the substrate selectivity of animal CXEs, such as porcine liver esterase (PLE) have been well studied, the respective enzymes in plants have yet to be defined and their activities determined. Using Arabidopsis thaliana (At) as a source, five representative members of the a/b hydrolase AtCXE family of proteins have been cloned, expressed and the purified recombinant proteins assayed for esterase activity with xenobiotic substrates. Two members, AtCXE5 and AtCXE18 were found to be active carboxylesterases, though AtCXE5 proved to be highly unstable as a soluble protein. AtCXE18 and the previously characterised S-formylglutathione hydrolase from Ara- bidopsis (AtSFGH) were assayed against a series of esters based on methylumbelliferone in which the acyl moiety was varied with respect to size and conformation. The same series was used to assay crude esterase preparation from Arabidopsis plants and the results com- pared with those obtained with the commonly used PLE. With straight chain esters, AtCXE18 behaved like PLE, but the Arabidopsis hydrolases proved less tolerant of branched chain acyl components than the mammalian enzyme. While none of the enzyme preparations accurately reflected all the activities determined with crude Arabidopsis protein extracts, the plant enzymes proved more useful than PLE in predicting the hydrolysis of the more sterically constrained esters. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Arabidopsis thaliana; Carboxylesterase; Ester series; a/b Hydrolases; Methylumbelliferone 1. Introduction Serine hydrolases are a diverse group of enzymes, which catalyse hydrolytic and transacylating reactions with both natural and synthetic substrates (Heikinheimo et al., 1999). In the case of drugs and pesticides, hydrolysis radi- cally alters biological activity and trans-membrane trans- port and so the respective hydrolases are key proteins in determining bioavailability and bioactivity (Satoh and Hosokawa, 1998). In microorganisms, the hydrolases active toward synthetic amides, carboxyesters and thioes- ters have been studied in some detail due to their impor- tance in the biodeterioration of polymers and the bioremediation of pollutants (Ro et al., 2004). Similarly, in mammals and insects these enzymes have attracted attention due to their roles in drug and insecticide metabo- lism, respectively (Satoh and Hosokawa, 1998; Oakeshott et al., 1999). In contrast, the corresponding enzymes in plants have received far less attention. This is surprising, given their importance in determining the uptake and bio- logical activity of important classes of herbicides, fungi- cides and insecticides (Cummins et al., 2001; Haslam et al., 2001). For example, insecticides such as the pyre- throids are detoxified by plant esterases, limiting their bio- availability in crop protection (Preiss et al., 1988). Esterases also bioactivate pro-herbicides to their phyto- toxic alcohols or acids, with the differential rates of hydro- lysis in different plants contributing to selective weed control (Cummins and Edwards, 2004; Haslam et al., 2001). The relationship between chemical structure and the rates of pesticide hydrolysis in plants would therefore 0031-9422/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2006.12.014 * Corresponding author. Tel.: +44 191 334 1318; fax: +44 191 334 1201. E-mail address: Robert.Edwards@durham.ac.uk (R. Edwards). www.elsevier.com/locate/phytochem Phytochemistry 68 (2007) 811–818 PHYTOCHEMISTRY