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.
Title: Selectivity of Rhizomucor miehei lipase as affected by choice of cosubstrate system in ester modification reactions in organic media Arsan J, Parkin KL Ref: Biotechnol Bioeng, 69:222, 2000 : PubMed
Fatty acid (FA) selectivity of immobilized Rhizomucor miehei lipase was determined for various cosubstrate systems for ester modification involving competing n-acyl-donor substrates of even-chain length (C4-C16; FA or their methyl esters, FAME) and either n-propanol or propyl acetate in hexane. Acyl-chain-length optima were observed for C8 and C14/16 in all cases. Upon changing between cosubstrate systems of [FA + propanol] to [FAME + propanol] to [FAME + propyl acetate], there was a general shift in selectivity toward shorter-chain-length FA (C4-C8). The greatest degree of reaction selectivity (based on ratios of selectivity constants) among the FA substrates was 3.1 for the [FA + propanol], 2.5 for the [FAME + propanol], and 1.4 for the [FAME + propyl acetate] cosubstrate systems. For esterification reactions between C6 FA and reactive members of a series of aliphatic and aromatic alcohols, the greatest degree of selectivity observed was 3.6.
