High-Resolution Crystal Structure of Plant Carboxylesterase AeCXE1, from Actinidia eriantha, and Its Complex with a High-Affinity Inhibitor Paraoxon
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- 作者：Nadeesha R. Ileperuma ; Sean D. G. Marshall ; Christopher J. Squire ; Heather M. Baker ; John G. Oakeshott ; Robyn J. Russell ; Kim M. Plummer ; Richard D. Newcomb ; Edward N. Baker
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：February 20, 2007
- 年：2007
- 卷：46
- 期：7
- 页码：1851 - 1859
- 全文大小：613K
- 年卷期：v.46,no.7(February 20, 2007)
- ISSN：1520-4995
文摘
Carboxylesterases (CXEs) are widely distributed in plants, where they have been implicatedin roles that include plant defense, plant development, and secondary metabolism. We have cloned,overexpressed, purified, and crystallized a carboxylesterase from the kiwifruit species Actinidia eriantha(AeCXE1). 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 
/
ddle">-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 ofcarboxylester 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-carbon acyl adduct bound to the active siteSer169 in the native structure. AeCXE1 was also found to be inhibited by organophosphates, with paraoxon(IC50 = 1.1 
M) a more potent inhibitor than dimethylchlorophosphate (DMCP; IC50 = 9.2 M). Thestructure of AeCXE1 with paraoxon 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 carboxylesterases in plants.
/ddle">-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 ofcarboxylester 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-carbon acyl adduct bound to the active siteSer169 in the native structure. AeCXE1 was also found to be inhibited by organophosphates, with paraoxon(IC50 = 1.1 M) a more potent inhibitor than dimethylchlorophosphate (DMCP; IC50 = 9.2 M). Thestructure of AeCXE1 with paraoxon 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 carboxylesterases in plants.