Journal Article

Structural basis for catalysis and selectivity of phospholipid synthesis by eukaryotic choline-phosphotransferase

Horibata, Yasuhiro, Kwarcinski, Frank E., Lam, Vinson, Tall, Gregory G., Hubbard, Akane, Raczkowski, Ashleigh M., White, Betsy, Roberts, Jacquelyn R., Ohi, Melanie D., Maeda, Shoji, Sugimoto, Hiroyuki

2025

Overview

Phospholipids are the most abundant component in lipid membranes and are essential for the structural and functional integrity of the cell. In eukaryotic cells, phospholipids are primarily synthesized de novo through the Kennedy pathway that involves multiple enzymatic processes. The terminal reaction is mediated by a group of cytidine-5′-diphosphate (CDP)-choline /CDP-ethanolamine-phosphotransferases (CPT/EPT) that use 1,2-diacylglycerol (DAG) and CDP-choline or CDP-ethanolamine to produce phosphatidylcholine (PC) or phosphatidylethanolamine (PE) that are the main phospholipids in eukaryotic cells. Here we present the structure of the yeast CPT1 in multiple substrate-bound states. Structural and functional analysis of these binding-sites reveal the critical residues for the DAG acyl-chain preference and the choline/ethanolamine selectivity. Additionally, we present the structure in complex with a potent inhibitor characterized in this study. The ensemble of structures allows us to propose the reaction mechanism for phospholipid biosynthesis by the family of CDP-alcohol phosphotransferases (CDP-APs). Here, the authors present the cryo-EM structure of yeast CPT1, a critical enzyme in phospholipid synthesis, identifying residues crucial for substrate preference. This enable a reaction mechanism for the family of CDP-alcohol phosphotransferases to be proposed.

• Publisher: Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
• Subject: 101/28 / 631/45/173 / 631/535/1258/1259 / 631/57/2271 / 82/58 / 82/80 / Alcohol / Binding Sites / Biocatalysis / Biosynthesis / Catalysis / Chemical synthesis / Choline / Crystallography, X-Ray / Cytidine Diphosphate - analogs & derivatives / Cytidine Diphosphate Choline - chemistry / Cytidine Diphosphate Choline - metabolism / Developmental biology / Diglycerides / Diglycerides - biosynthesis / Diglycerides - chemistry / Diglycerides - metabolism / Enzymes / Ethanolamine / Ethanolamines / Functional analysis / Humanities and Social Sciences / Interfaces / Lecithin / Lipid membranes / Lipids / Medicine / Membranes / Models, Molecular / multidisciplinary / Phosphatidylcholine / Phosphatidylcholines - biosynthesis / Phosphatidylcholines - chemistry / Phosphatidylcholines - metabolism / Phosphatidylethanolamine / Phosphatidylethanolamines - biosynthesis / Phosphatidylethanolamines - chemistry / Phosphatidylethanolamines - metabolism / Phospholipids / Phospholipids - biosynthesis / Phospholipids - chemistry / Phospholipids - metabolism / Phosphotransferase / Phosphotransferases (Alcohol Group Acceptor) - chemistry / Phosphotransferases (Alcohol Group Acceptor) - metabolism / Physiology / Preferences / Reaction mechanisms / Residues / Saccharomyces cerevisiae - metabolism / Saccharomyces cerevisiae Proteins - chemistry / Saccharomyces cerevisiae Proteins - genetics / Saccharomyces cerevisiae Proteins - metabolism / Science / Science (multidisciplinary) / Structure-function relationships / Substrate preferences / Substrate Specificity / Yeast