Catalytically important residues in the S. cerevisiae lysophospholipid acyltransferase, Lpt1p (605.15)

Members of the membrane bound o‐acyltransferase family mediate reactions in phospholipid and triglyceride synthesis, cholesterol esterification, and protein modification. Aside from a conserved histidine, the amino acids critical for catalysis in this family remain to be conclusively determined. Thus, the reaction mechanism remains undetermined. We used bioinformatics to identify residues highly conserved among homologs spanning three kingdoms and to perform site directed mutagenesis of the respective residues in the S. cerevisiae homolog, LPT1. Eight residues were identified which have R‐groups containing hydroxyl, carboxyl, and primary amine groups. Initial assays suggest that an acid / base mechanism is utilized by Lpt1p to esterify lysophosphatidylcholine with oleoyl‐CoA. To further examine Lpt1’s mechanism, utilization of three 18‐carbon monounsaturated acyl‐CoA species with differing double bond locations was assayed in vitro. Interestingly, location of the double bond at position Δ11 lowered the apparent Vmax by six fold and at position Δ6 lowered it two fold compared to the Δ9 species. No activity was detected with a 24‐carbon, Δ9 acyl‐CoA species. Together, these data suggest in Lpt1p, the acyl‐CoA substrate lies in a protein furrow, uniquely suited to accommodate acyl‐CoA species with less than 24 carbons and a double bond nine carbons from the thioester. Grant Funding Source : Supported by the University of Michigan Office of Research and Sponsored Programs