Mechanistic Insights into an Efficient Aqueous Ethyl Ester Synthase EsRP3 from <i>Rhizomucor pusillus</i> : Catalytic Properties and Thermostability

In this study, we discovered and characterized efficient aqueous ethyl-ester synthase EsRP3 with high catalytic activity toward medium-chain and aromatic acids. Under aqueous ethanol conditions (2 mol/L ethanol), EsRP3 reached maximal conversions of 36.2% for hexanoic acid (5 mmol/L), 77.6% for octanoic acid (25 mmol/L), and 50.7% for 3-phenylpropionic acid (25 mmol/L) and remained highly active from pH 3 to 6. Protonation states of His203 (binding) and His307 (catalysis) may modulate the pH-dependent catalytic efficiency. Combined molecular dynamics, electrostatic potential analysis, and site-directed mutagenesis indicated that lid-domain flexibility, a positively charged active-site environment, and strong fatty-acid binding contribute to the high aqueous esterification activity of EsRP3. Consistently, the Q209L mutant further enhanced catalytic activity likely by increasing lid-domain flexibility. For stability, mutational analysis revealed that internal hydrogen-bond networks play a critical role in thermal robustness. Overall, this study provides mechanistic insights into the catalytic efficiency and stability of aqueous ester synthases and may contribute to their engineering and potential applications in the food, chemical, and pharmaceutical industries.