Expression, purification, and <i>in vitro</i> characterization of the carboxylesterase CEST-9.2 from <i>Caenorhabditis elegans</i>

The nematode Caenorhabditis elegans biosynthesizes the ascarosides, a large, modular family of pheromones that are used in chemical communication. A number of carboxylesterase domain-containing (CEST) enzymes are responsible for decorating the glycolipid core of the ascarosides with a variety of modifications. However, these enzymes, which are homologous to human carboxylesterases and acetylcholinesterase, have not been characterized biochemically, and thus the mechanism whereby they attach different modifications to the ascarosides is unknown. Here, we report the expression, purification, and biochemical characterization of a soluble CEST enzyme for the first time. In this study, we focused on CEST-9.2, which is responsible for making (E)-2-methyl-2-butenoyl (MB)-modified ascarosides. We identified candidate substrates for the enzyme, and we successfully expressed a truncated version of CEST-9.2, which is lacking the transmembrane domain, in several expression systems, including Escherichia coli, Pichia pastoris, and Spodoptera frugiperda Sf9 cells. The purified CEST-9.2 from each of these systems was tested against candidate substrates, including ascarosides and either MB-coenzyme A (CoA), MB-choline, or MB-carnitine. No enzymatic activity was detected using these substrates, suggesting that either the transmembrane domain is necessary for activity or that the correct substrates have not yet been identified. We showed that the purified CEST-9.2 from Sf9 cells is well-folded and dimeric, offering a potential starting point for future structural and mechanistic studies.