Characterization and Mechanism Study of a Novel Ethanol Acetyltransferase from (EatH) with Good Thermostability, pH Stability, and Broad Alcohol Substrate Specificity.

Ethyl acetate, one of the most essential industrial compounds, has a broad range of applications, including flavors, fragrances, pharmaceuticals, cosmetics, and green solvents. Eat1 is accountable for bulk ethyl acetate production in yeasts, yet its properties and molecular mechanism are not well characterized. In this study, an gene from was obtained through gene mining. EatH showed the highest activity at pH 7.5 and 35 °C and preferred short-chain acyl substrates but had a broad alcohol substrate spectrum from short-chain primary alcohols to aromatic alcohols. Its and / values toward NPA were measured to be 1.16 mM and 29.03 L·mmol·s, respectively. The structure of EatH was composed of a lid domain and a core catalytic domain, with the catalytic triad of Ser124, Asp148, and His296. Additionally, crucial residues and their mechanism were analyzed through molecular docking, site-directed mutagenesis, and molecular dynamics simulation. The mutants N149A, N149K, and N149S showed enhanced enzyme activity toward NP-hexanoate to 5.0-, 6.6-, and 3.6-fold, and Y204S enhanced enzyme activity for NP-butyrate by 2.6 times via creating a wider substrate binding pocket and enhancing hydrophobicity. Collectively, this work provided a theoretical basis for the further rational design of EatH and enriched the understanding of the Eat family.