C
arboxylesterases (C
XEs)
are widely distributed in plants, where they have been implicatedin roles that include plant defense, plant development, and second
ary metabolism. We have cloned,overexpressed, purified, and crystallized a c
arboxylesterase from the kiwifruit species
Actinidia eriantha(AeCX
E1). The structure of AeCXE1 was determined by X-ray crystallography at 1.4 Å resolution. Thecrystal structure revealed that AeCXE1 is a member of the
![](/images/gifch<font color=)
ars/alpha.gif" BORDER=0>/
![](/images/gifch<font color=)
ars/beta2.gif" BORDER=0 ALIGN="middle">-hydrolase fold superfamily, most closelyrelated structurally to the hormone-sensitive lipase subgroup. The active site of the enzyme, located in an11 Å deep hydrophobic gorge, contains the conserved catalytic triad residues Ser169, Asp276, and His306.Kinetic analysis using
artificial ester substrates showed that the enzyme can hydrolyze a range ofc
arboxylester substrates with acyl groups ranging from C2 to C16, with a preference for butyryl moieties.This preference was supported by the discovery of a three-c
arbon acyl adduct bound to the active siteSer169 in the native structure. AeCXE1 was also found to be inhibited by organophosphates, with p
araoxon(IC
50 = 1.1
![](/images/entities/mgr.gif)
M) a more potent inhibitor than dimethylchlorophosphate (DMCP; IC
50 = 9.2
![](/images/entities/mgr.gif)
M). Thestructure of AeCXE1 with p
araoxon bound was determined at 2.3 Å resolution and revealed that theinhibitor binds covalently to the catalytic serine residue, with virtually no change in the structure of theenzyme. The structural information for AeCXE1 provides a basis for addressing the wider functionalroles of c
arboxylesterases in plants.