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 AC AT-related enzyme family, Are2p, as a model, we targeted phylogenetically c onserved sequences for mutagenesis in order to identify functionally import ant motifs. Deletion, truncation, and missense mutations implicate a regula tory role for the amino-terminal domain of Are2p and identified two carboxy l-terminal motifs as required for catalytic activity. A serine-to-leucine m utation in the (H/Y)SF motif (residues 338-340), unique to sterol esterific ation enzymes, nullified the activity and stability of yeast Are2p. Similar ly, a tyrosine-to-alanine change in the FYxDWWN motif of Are2p (residues 52 3-529) produced an enzyme with decreased activity and apparent affinity for oleoyl-CoA. Mutagenesis of the tryptophan residues in this motif completel y abolished activity. In human ACAT1, mutagenesis of the corresponding moti fs (residues 268-270, and 403-409, respectively) also nullified enzymatic a ctivity.ie On the basis of their critical roles in enzymatic activity and t heir sequence conservation, we propose that these motifs mediate sterol and acyl-CoA binding by this class of enzymes.