Cloning, expression, tertiary structure, activity characteristics, design of inhibitor, hydrolysis and inhibition mechanisms analysis of carp adipose triglyceride lipase (ATGL).

Lipid hydrolysis catalyzed by endogenous enzymes is a major factor reducing the quality of meat products. Adipose triglyceride lipase (ATGL) initiates triacylglycerol hydrolysis and is therefore a key enzyme in this process. In this study, the N-terminal domain of carp ATGL was cloned. Recombinant ATGL was successfully expressed and purified, and its activity was characterized at different temperatures and pH values. The predicted tertiary structure of ATGL was analyzed. Carp ATGL is a monomeric protein composed of nine α-helices and seven β-strands, with a radius of gyration (Rg) of 1.84 nm. Its active center is a catalytic dyad (Ser47-Asp166) located at the bottom of an active groove that is predominantly hydrophobic. The active groove is surrounded by three flexible and hydrophobic loops, which are involved in binding hydrophobic lipids. The hydrolysis mechanism of ATGL was then elucidated. Finally, an inhibitor of ATGL was designed: a small peptide consisting of hydrophobic essential amino acids (Lys-Leu-Ile-Met-Val). After the addition of the small peptide, lipid hydrolysis and oxidation in carp surimi during storage were effectively controlled. And this peptide can be misrecognized by the hydrophobic active groove of ATGL and forms a steric hindrance, thereby preventing triacylglycerol binding.