Abstract:
Esters are a ubiquitous group of secondary metabolites found in plants. Their association with a wide range of important plant processes has made esters and their metabolism the target of a large number of studies. We selected two enzyme families involved in ester biosynthesis, the alcohol acyl transferases (AATs) and the Carboxylesterases (CXEs) as targets for structural and functional studies. Three members of the AAT family from apple Mahis pumila, MpAATl, MpAT4 and MpAT6 were chosen as targets. A major hurdle was faced during the overexpression and purification ofMpAATl, in which the enzyme co-purified with the E.coli chaperone protein, GroEL. Changing the E.coli host cells used for protein overexpression from C43(DE3) cells to Rosetta gami 2(De3)pLysS cells led to successful overexpression of MpAATl without GroEL and soluble overexpression of previously insoluble MpAT4 and MpAT6. The enzymes were purified and used for crystallization studies. Suitable crystallization conditions for any of these enzymes have not been identified at this stage. The AeCXEl enzyme from kiwifruit Actinidia eriantha was the main focus of study from the CXE family. This enzyme was successfully overexpressed, purified and crystallized. The structure of AeCXEl was determined by X-ray crystallography at 1.4 A resolution. The crystal structure revealed that AeCXEl is a member of the α∕β hydrolase fold superfamily. Ligand docking studies provided a structural basis for the substrate preferences displayed by AeCXEl. The Carboxylesterase activity of AeCXEl was inhibited by the organophosphates paraoxon and dimethylchlorophosphate (DMCP), with paraoxon being the more potent inhibitor. The structure of AeCXEl in complex with paraoxon was determined at 2.3 A defining the mode of AeCXEl inhibition by paraoxon. Three observations made during this study: the ability of the AeCXEl active site to accommodate a wide range of ester and non-ester substrates, the broad expression pattern displayed by the gene encoding the AeCXEl ortholog from Actinidia chinensis and the ability of AeCXEl to hydrolyze the lipid substrate tributyrin all suggest a general rather than a specific role for AeCXEl in plants. Attempts were made to obtain a structural basis for substrate preferences by the CXEs, AtCXE18, AtCXE20 and MpCXEl, findings of which need to be clarified by further studies.