Biochemical Characterization of ABHD14A, an Outlying Member of the Metabolic Serine Hydrolase Family

Abstract

Certain uncharacterized 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 can turn over short-chain esters. Notably, ABHD14A exhibits a coenzyme A-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 the subcellular localization of ABHD14A, heterologous overexpression of an ABHD14A-GFP fusion variant in HEK293T cells was performed. Here, we found that the ABHD14A-GFP fusion protein is catalytically active and localizes specifically to the Golgi apparatus, suggesting a specialized role in the secretory pathway biology. Together, these findings establish the enzymatic activity, mechanistic features, and subcellular localization of ABHD14A while providing essential biochemical tools that can now enable the systematic discovery of its physiological substrates and regulatory contexts.