Saccharomyces cerevisiae Atf1p is an alcohol acetyltransferase and a thioesterase in vitro.
The alcohol-O-acyltransferases are bisubstrate enzymes that catalyse the transfer of acyl chains from an acyl-coenzyme A (CoA) donor to an acceptor alcohol. In the industrial yeast Saccharomyces cerevisiae this reaction produces acyl esters that are an important influence on the flavour 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 characterize Atf1p, the major alcohol acetyltransferase from S. 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 thioesterase 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. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd.
School of Biochemistry, University of Bristol, Bristol, UK.
Bethany
B
Nancolas
School of Chemistry, University of Bristol, Bristol, UK.
Ian D
ID
Bull
School of Biochemistry, University of Bristol, Bristol, UK.|Bristol Centre for Functional Nanomaterials, University of Bristol, Bristol, UK.
Richard
R
Stenner
School of Biochemistry, University of Bristol, Bristol, UK.|BrisSynBio, Life Sciences Building, Tyndall Avenue, Bristol, UK.
Virginie
0000-0003-2912-056X
V
Dufour
School of Biochemistry, University of Bristol, Bristol, UK.|BrisSynBio, Life Sciences Building, Tyndall Avenue, Bristol, UK.
Paul
0000-0001-9889-0248
P
Curnow
D000123:Acetyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D008401:Gas Chromatography-Mass Spectrometry; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification / Q000378:metabolism*
D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D000123:Acetyltransferases; C079238:alcohol O-acetyltransferase
enzymes; metabolism; protein purification