Biochemical characterization and mutational analysis of lysophosphatidic acid acyltransferases of Escherichia coli highlighting their involvement in the generation of membrane phospholipid diversity.

Lysophosphatidic acid acyltransferase (LPAAT) is an enzyme responsible for the second acylation step of phospholipid biosynthesis and transforms lysophosphatidic acid to phosphatidic acid, a universal precursor of various phospholipids. In addition to the well-studied plsC-encoded LPAAT (EcPlsC), we previously found that Escherichia coli has another LPAAT that is encoded by yihG (EcYihG). EcPlsC and EcYihG are integral membrane proteins and have never been solubilized and purified in their active form. To better understand the difference in their enzymatic functions and how the two paralogs differently contribute to lipid diversity, we established a method to purify both enzymes in their active form and comparatively analyzed their biochemical characteristics. Our findings illustrate that EcPlsC possesses the highest activity at pH 8.0 and 37 °C with selectivity for unsaturated fatty acyl-CoAs (e.g. palmitoleoyl-CoA), whereas EcYihG works optimally at pH 7.5 and 30 °C and prefers saturated fatty acyl-CoAs (e.g. myristoyl-CoA). In addition, we performed a mutational analysis based on AlphaFold2 models and revealed that one residue, which is located at the putative acyl-donor-selectivity tunnel entrance, plays a pivotal role in selecting acyl donor substrates. This provides new insights into how LPAATs recognize specific fatty acyl groups and incorporate them into membrane phospholipids.