title: The yeast enzyme Eht1 is an octanoyl-CoA:ethanol acyltransferase that also functions as a thioesterase.
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 acid ethyl esters are important flavour 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 at 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 k(cat)  = 0.28 ± 0.02/s and K(M)  = 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.
authors.affiliation: School of Biochemistry, University of Bristol, UK.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Knight
authors.forename: Ian D
authors.initials: ID
authors.lastname: Bull
authors.forename: Paul
authors.initials: P
authors.lastname: Curnow
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism*; D000217:Acyltransferases / Q000378:metabolism*; D004252:DNA Mutational Analysis; D015870:Gene Expression; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013869:Thiolester Hydrolases / Q000378:metabolism*
chemical_list: D000214:Acyl Coenzyme A; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C034681:octanoyl-coenzyme A; D000217:Acyltransferases; C000588861:EHT1 protein, S cerevisiae; C020124:ethanol acyltransferase; D013869:Thiolester Hydrolases
keywords: coenzyme A; enzyme kinetics; medium chain fatty acid ethyl ester; protein expression; protein purification