Figure 12.

Site-directed mutagenesis of acyltransferase and lipase motifs. A, The MGAT assay was performed for 15 min with [¹⁴C]MAG and 20 µm oleoyl-CoA using wild-type and HX₄D mutant recombinant AhMGAT proteins: H62A, His-62 was replaced with Ala; D67A, Asp-67 was replaced with Ala; H62AX4D67A, double mutant, His-62 and Asp-67 were replaced with Ala; S97A, Ser-97 was replaced with Ala; S138A, Ser-138 was replaced with Ala; wild-type AhMGAT. FFA, Free fatty acid. B, The MAG hydrolase assay was performed with 50 μm [¹⁴C]MAG using wild-type and mutant recombinant AhMGAT proteins. The reaction was stopped by extracting lipids, and the lipids were separated on a silica-TLC plate using petroleum ether:diethyl ether:acetic acid (70:30:1, v/v) as the solvent system. Lane −E, No-enzyme control; lane B, enzyme fraction was boiled for 5 min, and assay was performed. Values are means ± sd of three independent experiments. C, The LPC hydrolase assay was performed with 50 μm [¹⁴C]LPC using wild-type and mutant recombinant AhMGAT proteins. The reaction was stopped, and lipids were resolved on a TLC plate using chloroform:methanol:28% ammonia (65:25:5, v/v) as the solvent system. Results are represented as percentage means ± sd of three independent experiments.