The yeast enzyme Eht1 is an octanoyl-CoA:ethanol acyltransferase that also functions as a thioesterase

Michael J Knight, Ian D Bull, Paul Curnow

Research output: Contribution to journalArticle (Academic Journal)

17 Citations (Scopus)

Abstract

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.
Original languageEnglish
JournalYeast
Early online date13 Oct 2014
DOIs
Publication statusPublished - 2014

Keywords

  • Enzyme kinetics
  • protein purification
  • medium chain fatty acid ethyl ester
  • Coenzyme A

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