title: High-resolution crystal structure of plant carboxylesterase AeCXE1, from Actinidia eriantha, and its complex with a high-affinity inhibitor paraoxon.
abstract: Carboxylesterases (CXEs) are widely distributed in plants, where they have been implicated in 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 A resolution. The crystal structure revealed that AeCXE1 is a member of the alpha/beta-hydrolase fold superfamily, most closely related structurally to the hormone-sensitive lipase subgroup. The active site of the enzyme, located in an 11 A 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 of carboxylester 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 site Ser169 in the native structure. AeCXE1 was also found to be inhibited by organophosphates, with paraoxon (IC50 = 1.1 muM) a more potent inhibitor than dimethylchlorophosphate (DMCP; IC50 = 9.2 muM). The structure of AeCXE1 with paraoxon bound was determined at 2.3 A resolution and revealed that the inhibitor binds covalently to the catalytic serine residue, with virtually no change in the structure of the enzyme. The structural information for AeCXE1 provides a basis for addressing the wider functional roles of carboxylesterases in plants.
authors.affiliation: School of Biological Sciences, University of Auckland, Auckland, New Zealand.
authors.forename: Nadeesha R
authors.initials: NR
authors.lastname: Ileperuma
authors.forename: Sean D G
authors.initials: SD
authors.lastname: Marshall
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Squire
authors.forename: Heather M
authors.initials: HM
authors.lastname: Baker
authors.forename: John G
authors.initials: JG
authors.lastname: Oakeshott
authors.forename: Robyn J
authors.initials: RJ
authors.lastname: Russell
authors.forename: Kim M
authors.initials: KM
authors.lastname: Plummer
authors.forename: Richard D
authors.initials: RD
authors.lastname: Newcomb
authors.forename: Edward N
authors.initials: EN
authors.lastname: Baker
mesh_terms: D029042:Actinidia / Q000201:enzymology*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002265:Carboxylic Ester Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004952:Esters / Q000737:chemistry; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010261:Paraoxon / Q000737:chemistry*; D010940:Plant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D016415:Sequence Alignment
chemical_list: D004952:Esters; D010940:Plant Proteins; D002265:Carboxylic Ester Hydrolases; D010261:Paraoxon