Biochemical characterization of ABHD14A, an outlying member of the metabolic serine hydrolase family

Certain uncharacterised members of the metabolic serine hydrolase enzyme family remain difficult to annotate due to poor tractability, context-dependent expression, and the absence of defined biochemical activities. Here, we provide the first functional characterization of the human enzyme ABHD14A. By engineering a soluble N-terminally truncated variant, we demonstrate by gel-based activity-based protein profiling and p-nitrophenyl-ester hydrolysis assays that ABHD14A is an active enzyme that preferentially turns over short-chain esters. Notably, ABHD14A exhibits a CoA-dependent enhancement of p-nitrophenyl-acetate hydrolysis, indicative of a ping-pong type acetyltransferase mechanism similar to that previously described for another homologous ABHD14-enzyme, ABHD14B. To investigate endogenous protein levels, we generated a high-affinity polyclonal antibody and found that ABHD14A is undetectable across a panel of immortalized mammalian cell lines and adult mouse tissues, challenging current transcriptomic database predictions. Upon heterologous expression in HEK293T cells, full length ABHD14A is catalytically active and localizes specifically to the Golgi apparatus, suggesting a specialized role in secretory pathway biology. Together, these findings establish the enzymatic activity, mechanistic features, and subcellular localization of ABHD14A, while providing essential biochemical and immunological tools that now enable the systematic discovery of its physiological substrates and regulatory contexts.