Structure-based mechanism for CDP-diglyceride hydrolase, CdhA from Escherichia coli

Abstract

CDP-diglyceride is an essential liponucleotide precursor in bacterial phospholipid synthesis, serves as a donor of phosphatidyl residues. Its synthesis and utilization are tightly regulated by various enzymes, including CdhA, which catalyzes the hydrolysis of CDP-diglyceride to modulate phospholipid biosynthesis. However, prior to this study, only the apo structure of E. coli CdhA had been resolved, with no further experimental validation of its catalytic mechanism. In this study, we present the X-ray crystal structure of E. coli CdhA complexed with a cytidine monophosphate (CMP), a hydrolysis reaction product. The structure reveals CMP binding within a negatively charged groove containing highly conserved histidine residues, His140 and His142, in the putative HIT domain. In vitro hydrolysis assays of single amino acid substitution mutants validate the essential roles of these conserved histidines and basic residues around CMP in CdhA’s catalytic activity. Additionally, the presence of hydrophobic residues near the active site suggest fatty acid chains of CDP-diacylglyceride may interact with these nonpolar residues, thereby facilitating substrate binding and enhancing specificity. These findings provide structural basis mechanism of CDP-diglyceride hydrolysis and advance our understanding of bacterial phospholipid metabolism.