Fatty acid ethyl esters are secondary metabolites that are produced during microbial fermentation, in fruiting plants, and in higher organisms during ethanol stress. In particular, volatile medium-chain fatty esters are important flavor compounds that impart desirable fruit aromas to fermented beverages including beer and wine. The biochemical synthesis of medium-chain fatty acid ethyl esters is poorly understood but likely involves acyl-CoA:ethanol O-acyltransferases. Here, we characterize the enzyme ethanol hexanoyl transferase 1 (Eht1) from the brewer’s yeast Saccharomyces cerevisiae. Full-length Eht1 was successfully overexpressed from a recombinant yeast plasmid and purified on the milligram scale after detergent solubilization of sedimenting membranes. Recombinant Eht1 was functional as an acyltransferase and, unexpectedly, was optimally active toward octanoyl-CoA with kcat = 0.28 ± 0.02 s-1 and KM = 1.9 ± 0.6 μM. Eht1 was also revealed to be active as a thioesterase, but was not able to hydrolyse p-nitrophenyl acyl esters in contrast to the findings of a previous study. Low-resolution structural data and site-directed mutagenesis provide experimental support for a predicted α/β-hydrolase domain featuring a Ser-Asp-His catalytic triad. The S. cerevisiae gene YBR177C/EHT1 should thus be reannotated as coding for an octanoyl-CoA:ethanol acyltransferase that can also function as a thioesterase.
- Enzyme kinetics
- protein purification
- medium chain fatty acid ethyl ester
- Coenzyme A