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Journal of Lipid Research, Vol. 42, 1282-1291, August 2001
Copyright © 2001 by Lipid Research, Inc.

Original Article

Identification of potential substrate-binding sites in yeast and human acyl-CoA sterol acyltransferases by mutagenesis of conserved sequences

Correspondence to: Stephen L. Sturley, To whom correspondence should be addressed., sls37{at}columbia.edu (E-mail)

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., D. Cromley, J. T. Billheimer, and S. L. Sturley. Identification of potential substrate-binding sites in yeast and human acyl-CoA sterol acyltransferases by mutagenesis of conserved sequences. J. Lipid Res. 2001. 42: 1282;–1291.

Supplementary key words: ACAT cholesterol, steryl ester

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