Saccharomyces cerevisiae Atf1p is an alcohol acetyltransferase and a thioesterase in vitro

Bethany Nancolas, Ian Bull, Richard Stenner, Virginie Dufour, Paul Curnow

Research output: Contribution to journalArticle (Academic Journal)

11 Citations (Scopus)
409 Downloads (Pure)

Abstract

The alcohol-O-acyltransferases are bisubstrate enzymes that catalyse the transfer of acyl chains from an acyl-CoA donor to an acceptor alcohol. In the industrial yeast Saccharomyces cerevisiaethis reaction produces acyl esters that are an important influence on the flavor of fermented beverages and foods. There is also a growing interest in using acyltransferases to produce bulk quantities of acyl esters in engineered microbial cell factories. However, the structure and function of the alcohol-O-acyltransferases remain only partly understood. Here, we recombinantly express, purify and characterise Atf1p, the major alcohol acetyltransferase from Saccharomyces cerevisiae. We find that Atf1p is promiscuous with regard to the alcohol cosubstrate but that the acyltransfer activity is specific for acetyl-CoA. Additionally, we find that Atf1p is an efficient thioesterase in vitro with specificity towards medium chain-length acyl-CoAs. Unexpectedly, we also find that mutating the supposed catalytic histidine (H191) within the conserved HXXXDG active site motif only moderately reduces the activity of Atf1p. Our results imply a role for Atf1p in CoA homeostasis and suggest that engineering Atf1p to reduce the thioesterase activity could improve product yields of acetate esters from cellular factories.
Original languageEnglish
Pages (from-to)239-251
Number of pages13
JournalYeast
Volume34
Issue number6
Early online date6 Mar 2017
DOIs
Publication statusPublished - Jun 2017

Structured keywords

  • BrisSynBio
  • Bristol BioDesign Institute

Keywords

  • AAT
  • alcohol-O-acetyltransferase
  • CD, circular dichroism
  • CMC, detergent critical micelle concentration
  • CoA, Coenzyme A
  • GC-MS, Gas chromatography-mass spectrometry
  • SEC, size exclusion chromatography
  • Synthetic Biology

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