Identification of potential substrate-binding sites in yeast and human acyl-CoA sterol acyltransferases by mutagenesis of conserved sequences.
In mammals, the esterification of sterols by ACAT plays a critical role in eukaryotic lipid homeostasis. Using the predominant isoform of the yeast ACAT-related enzyme family, Are2p, as a model, we targeted phylogenetically conserved sequences for mutagenesis in order to identify functionally important motifs. Deletion, truncation, and missense mutations implicate a regulatory role for the amino-terminal domain of Are2p and identified two carboxyl-terminal motifs as required for catalytic activity. A serine-to-leucine mutation in the (H/Y)SF motif (residues 338-340), unique to sterol esterification enzymes, nullified the activity and stability of yeast Are2p. Similarly, a tyrosine-to-alanine change in the FYxDWWN motif of Are2p (residues 523-529) produced an enzyme with decreased activity and apparent affinity for oleoyl-CoA. Mutagenesis of the tryptophan residues in this motif completely abolished activity. In human ACAT1, mutagenesis of the corresponding motifs (residues 268-270, and 403-409, respectively) also nullified enzymatic activity. On the basis of their critical roles in enzymatic activity and their sequence conservation, we propose that these motifs mediate sterol and acyl-CoA binding by this class of enzymes.
Guo
Z
Z
Institute of Human Nutrition, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.
Cromley
D
D
Billheimer
J T
JT
Sturley
S L
SL
D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D017124:Conserved Sequence; D004951:Esterification; D017353:Gene Deletion; D015870:Gene Expression; D006801:Humans; D007930:Leucine / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D012694:Serine / Q000235:genetics; D002785:Sterol O-Acyltransferase; D013329:Structure-Activity Relationship; D014162:Transfection; D014364:Tryptophan / Q000235:genetics; D014443:Tyrosine / Q000235:genetics
D029701:Saccharomyces cerevisiae Proteins; D014443:Tyrosine; D012694:Serine; D014364:Tryptophan; D000217:Acyltransferases; C543388:ARE2 protein, S cerevisiae; D002785:Sterol O-Acyltransferase; D007930:Leucine; D000409:Alanine