ACYL CHAIN REMODELING BY LYSOPHOSPHOLIPID ACYLTRANSFERASE 3: POSITIONAL SELECTIVITY DETERMINANTS FOR ARACHIDONOYL-COA INCORPORATION AT THE SN-2 POSITION
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Objective: To define the molecular and structural determinants that confer LPCAT3 selectivity for arachidonoyl-CoA and its preferential incorporation at the sn-2 position of lysophospholipids. Methods: Human LPCAT3 was expressed in mammalian cells, and membrane fractions were used for steady-state kinetic analyses with 1- and 2-acyl-lysoPC and multiple acyl-CoA donors. Structure-guided mutagenesis targeted tunnel and side-pocket residues (L217A, F265A, L217A/F265A, H376A). Docking and molecular dynamics were performed on wild-type and mutant LPCAT3. LPCAT3-deficient cells were complemented with selected variants and analyzed by isotope-tracing lipidomics and functional readouts. Results: Wild-type LPCAT3 showed maximal catalytic efficiency for arachidonoyl-CoA (Km 5.2 ± 0.8 µM, kcat/Km set as 1.0) and lower efficiencies for 22:6-, 18:1- and 16:0-CoA (relative kcat/Km 0.70, 0.40, and 0.25, respectively). Initial rates toward 1-acyl-lysoPC were ~5-fold higher than for 2-acyl-lysoPC, with 92 ± 3% of incorporated arachidonate residing at sn-2. Mutations L217A, F265A, and L217A/F265A increased Km for arachidonoyl-CoA up to ~25 µM and reduced relative kcat/Km to 0.10, while decreasing sn-2 enrichment to ~55–65%. In LPCAT3-deficient cells, total arachidonoyl-PC rose from 0.11 ± 0.02 (vector) to 0.41 ± 0.04 (wild-type) but only to 0.17–0.24 with tunnel/side-pocket mutants. Novelty: The acyl-CoA tunnel and hydrophobic side pocket of LPCAT3 serves as a structural groove of the kinked arachidonoyl chain to bind it with high affinity and specific sn-2-positioning. Perturbation of this architecture reduces arachidonoyl-PC development and downstream lipid-signalling ability, which makes it clear that LPCAT3 is a mechanistic crossroads of regulating membrane PUFA content and arachidonate-dependent signalling.
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