Structural mechanism of membrane-associated cytidine diphosphate diacylglycerol diphosphatase in Escherichia coli

Abstract

Cytidine diphosphate diacylglycerol (CDP-DAG) diphosphatase (Cdh) regulates phospholipid biosynthesis by hydrolyzing CDP-DAG into cytidine monophosphate (CMP) and phosphatidic acid (PA), thereby maintaining the steady-state cellular levels of these key metabolic intermediates. Although CDP-DAG serves as a universal branch point in phospholipid metabolism across all three domains of life, Cdh is found predominantly in prokaryotes and, to a lesser extent, in eukaryotes. In Escherichia coli, Cdh is a membrane-associated enzyme belonging to the histidine-triad (HIT)-like hydrolase family and functions independently of metal ions. Here, we report two X-ray crystal structures of E. coli Cdh in complex with a reaction product, CMP, and an inhibitor, AMP, revealing the molecular basis of nucleotide recognition and substrate binding. Integrating structural and biochemical analyses, we identify a pair of conserved histidine residues, His-140 and His-142, as key catalytic determinants. Furthermore, we demonstrate that Cdh adopts a bitopic membrane topology, in which an N-terminal transmembrane helix spans the lipid bilayer and serves as the primary membrane anchor, positioning the catalytic domain at the membrane interface. Together, these findings establish Cdh as a monomeric, membrane-embedded HIT-like hydrolase and provide mechanistic insight into CDP-DAG turnover at the membrane-cytosol interface.