Characterization of the Plant CXE Carboxylesterase Family
A large family of plant carboxylesterase genes (CXEs) has been identified, which may encode enzymes involved in secondary metabolism (among other potential functions) in higher plants. CXE family members were identified from the model plants Arabidopsis thaliana (20) and Oryza sativa (48), from the commercial fruit crops Actinidia spp. (12), Malus pumila (12), and Vaccinium corymbosum (1), plus several CXEs previously identified from a variety of other plant species. Plant CXEs share conserved sequence motifs and secondary structure characteristics with members of the α/β hydrolase fold superfamily. Phylogenetic analysis of the plant CXE genes distinguishes eight distinct clades. Detailed examination of the A. thaliana genome reveals that the AtCXEs are widely distributed across the genome, being present in four out of five chromosomes. Of the inter-chromosomal duplication events, two have been mediated through newly identified partial chromosomal duplication events that also include other genes surrounding the AtCXE loci. Eighteen of the 20 AtCXE genes are expressed over a broad range of tissues, while the remaining two unrelated genes are expressed only in the flowers and siliques. Four plant CXE carboxylesterases (A. thaliana CXE18, 20, Actinidia spp. CXE1, and M. pumila CXE1), representing two separate phylogenetic clades (clades II and IV), were recombinantly expressed in bacteria. The expressed CXEs were inhibited by organophosphates. Kinetic analysis with a series of 4-methyl umbelliferyl esters showed that substrate affinity for all four CXEs increases as the acyl chain length increases. Reaction efficiency of the plant CXEs is split along clade boundaries, with the two clade II enzymes preferring short acyl chain esters (acetate to butyrate), while the two clade lV enzymes prefer mid to longer acyl chain esters (heptanoate to laurate). A large number of volatile esters commonly found in plants can be hydrolyzed by these CXEs. Substrates utilized range from simple straight chain hydrocarbons (e.g. butyl acetate, hexyl hexanoate) to esters possessing more complex branched (e.g. linalyl acetate) and aromatic structures (e.g. methyl salicylate), suggestive of a spacious active site. Preliminary metabolic profiling of four AtCXE T-DNA insertion mutants reveals subtle differences in metabolite levels when compared to wildtype plants. This initial characterization of the CXE family provides a foundation for future structural studies as well as hypotheses for the role(s) these enzymes play in plants.
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