Active site determinants of yeast Pah1 phosphatidate phosphatase activity and cellular functions

In the yeast Saccharomyces cerevisiae, the PAH1-encoded phosphatidate (PA) phosphatase plays a major role in the control of diacylglycerol and PA, which are crucial for the synthesis of the storage lipid triacylglycerol and membrane phospholipids as well as for diverse cellular processes. The catalytic core of Pah1 contains the haloacid dehalogenase-like domain, a catalytic domain in diverse phosphatases with the conserved motifs I-IV. In this work, we found the four active site motifs in Pah1 by sequence alignment and AlphaFold modeling and identified Arg-445 as an additional residue conserved in motif II of Pah1 and its orthologs. Mutational analyses of the Pah1 active site motifs showed that the conserved residues (Asp-398 and Asp-400 in motif I, Thr-443 and Arg-445 in motif II, Lys-496 in motif III, and Gly-529, Asn-530, and Asp-534 in motif IV) are essential for PA phosphatase (PAP) activity and the related cellular functions of the enzyme. The limited proteolysis analysis of unphosphorylated Pah1, which mimics the functional dephosphorylated form of Pah1, indicates that its overall structure is not affected by the active site mutations. In the liposome-binding assay, the active site mutations in Pah1 did not affect its association with the membrane. These findings demonstrate that the active site motifs are essential for Pah1 PAP activity and provide a mechanistic basis for lipid-associated diseases caused by mutations in the human lipin PAP.