Journal
JOURNAL OF LIPID RESEARCH
Volume 56, Issue 11, Pages 2143-2150Publisher
ELSEVIER
DOI: 10.1194/jlr.M062141
Keywords
enzymology; enzyme mechanisms; phospholipids; biosynthesis; phospholipids; metabolism; fatty acid; transferase; acyl-CoA diacylglycerol acyltransferase; active site; reaction mechanism; membrane bound O-acyltransferase
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Funding
- Undergraduate Fellowship - University of Michigan-Dearborn Office of Research and Sponsored Programs
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The esterification of lysophospholipids contributes to phospholipid synthesis, remodeling, and scavenging. Acyl-CoA-dependent lysophospholipid acyltransferase activity with broad substrate use is mediated by Saccharomyces cerevisiae Lpt1p. We sought to identify Lpt1p active site amino acids besides the histidine conserved among homologs and repeatedly found to be required for catalysis. In vitro Lpt1p assays with amino acid modifying agents implicated aspartate, glutamate, and lysine as active site residues. Threonine and tyrosine were not ruled out. Aligning the primary structures of functionally characterized LPT1 homologs from fungi, plants, and animals identified 11 conserved aspartate, glutamate, lysine, threonine, and tyrosine residues. Site-directed mutagenesis of the respective codons showed that changing D146 and E297 abolished activity without abolishing protein expression. The mechanism of Lpt1p was further analyzed using monounsaturated acyl-CoA species with different double bond positions. Delta 6 species showed the highest catalytic efficiency. We propose that D146 and E297 act in conjunction with H382 as nucleophiles that attack the hydroxyl group in lysophospholipids in a general acid/base mechanism. This sequential mechanism provides a precedent for other members of the membrane bound O-acyltransferase family. Also, Lpt1p optimally orients acyl-CoA substrates with 7.5 angstrom between a double bond and the thioester bond.
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