Deciphering the active site: Why motif conservation in Pah1 matters for yeast lipid homeostasis

The enzyme Pah1 is a central figure in the lipid metabolism of Saccharomyces cerevisiae, acting as a Mg²⁺-dependent phosphatidate (PA) phosphatase that converts PA into diacylglycerol. This catalytic step is vital for balancing the synthesis of storage lipids like triacylglycerol and the formation of membrane phospholipids. Pah1 is a member of the haloacid dehalogenase (HAD)-like superfamily, which is characterized by a conserved catalytic structure known as the Rossmann-like fold.

In a recent study published in JBC, Geordan Stukey (now in the Dominguez lab at U. Penn) and colleagues in the Carman lab at Rutgers-New Brunswick identified four specific active site motifs within the HAD-like domain of Pah1 through sequence alignment and AlphaFold modeling. These motifs (I–IV) were found to be essential for the enzyme's phosphatase activity and its various roles within the cell. The research highlighted several key residues, including 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. Notably, Arg-445 was identified as a residue conserved specifically in Pah1 and its orthologs but absent in other HAD-like enzymes, suggesting it may play a specialized role in lipid catalysis.

Stukey et al. employed mutational analyses to test the importance of these conserved residues. Mutations at these sites resulted in an almost complete loss of PA phosphatase activity. Interestingly, limited proteolysis and liposome-binding assays indicated that while catalytic activity was destroyed, the overall protein structure and membrane association capabilities remained largely unaffected. This suggests that these residues are specifically required for the phosphoryl transfer reaction rather than for maintaining the enzyme's physical stability or its recruitment to the nuclear/ER membrane.

The physiological consequences of these active site mutations are striking and mirror the phenotypes observed in Pah1-deficient (pah1Δ) yeast strains. Cells expressing the mutant alleles failed to restore triacylglycerol levels and showed a corresponding increase in membrane phospholipids. This lipid imbalance leads to a variety of deleterious effects, including the aberrant expansion of the nuclear/ER membrane and a significant reduction in the number of cytoplasmic lipid droplets. Furthermore, these cells exhibited growth defects at high temperatures or on nonfermentable carbon sources like glycerol. The loss of activity also prevented the normal repression of phospholipid biosynthetic genes, such as the CHO1-encoded phosphatidylserine synthase.

Beyond the world of yeast microbiology, these findings have significant implications for understanding human health. Pah1 is orthologous to the human lipin proteins, which are also Mg²⁺-dependent phosphatidate phosphatases. Dysfunction in human lipins is implicated in a range of lipid-related disorders, including lipodystrophy, type 2 diabetes, and rhabdomyolysis. The study specifically notes that the Thr-443 and Arg-445 residues in Pah1 correspond to residues in human lipin 1 and lipin 2 where mutations are known to cause Majeed syndrome and rhabdomyolysis.

Ultimately, this research provides a comprehensive mechanistic basis for how specific active site residues govern lipid synthesis and cellular physiology. By using Saccharomyces cerevisiae as a model organism, the authors have deepened our understanding of the HAD-like domain's function and the broader consequences of lipid metabolic dysfunction. This study reinforces the value of yeast as a powerful tool for investigating the molecular roots of human genetic diseases.

Stukey GJ, Sharma PK, Jog R, Kwiatek JM, Han GS, Carman GM. Active site determinants of yeast Pah1 phosphatidate phosphatase activity and cellular functions. J Biol Chem. 2025 Aug;301(8):110492. doi: 10.1016/j.jbc.2025.110492. Epub 2025 Jul 17. PMID: 40680843; PMCID: PMC12357310. https://www.jbc.org/article/S0021-9258(25)02342-7/fulltext