Abstract 1381 Identification of three catalytic motifs in the HAD-like domain from Saccharomyces cerevisiae phosphatidate phosphatase Pah1

In the model eukaryote Saccharomyces cerevisiae, the phosphatidate (PA) phosphatase Pah1 catalyzes the dephosphorylation of PA to produce diacylglycerol. The generated diacylglycerol serves as the immediate precursor for triacylglycerol synthesis, or it can be used to produce the membrane phospholipids phosphatidylcholine and phosphatidylethanolamine. Pah1 is a member of the evolutionarily ancient haloacid dehalogenase-like (HAD-like) enzyme superfamily. The conserved N-LIP and HAD-like domains in Pah1 are disparately located in the primary structure but are expected to co-fold in the tertiary structure as both are required for catalysis. Although the HAD-like domain contains the canonical DXDX(T/V) catalytic motif I needed for activity, the architecture of the active site has yet to be carefully examined. Utilizing structural predictive algorithms, three additional motifs within the HAD-like domain of Pah1 were identified based on the orientation of the amino acids in the active site when compared to other solved HAD protein structures. Amino acids Thr-443, Arg-445, Lys-496, Asn-530, and Asp-534 were identified as putative components of catalytic motifs II-IV conserved in HAD-like enzymes. Substitution mutants were constructed at each of the five residues and, when expressed in a pah1Δ mutant, were unable to fully complement the loss of PA phosphatase function within the cells. Phenotypes associated with a total loss of PA phosphatase function were seen with T443A, K496A, and N530A mutations while partial loss of function were seen in cells harboring R445A and D534A mutations. This work identifies three new catalytic motifs in the HAD-like domain of Pah1 and demonstrates that the essential N-LIP domain does not contribute to PA phosphatase activity through providing the conserved HAD phosphatase motifs. Supported by NIH grant 136128