Structural insights into substrate binding, residue contributions, and catalytic mechanism of phosphopantetheine adenylyltransferase from Helicobacter pylori.

Abstract

Phosphopantetheine adenylyltransferase (PPAT) (PPAT; EC 2.7.3.3) is a key enzyme in coenzyme A (CoA) biosynthesis. It catalyzes the reversible transfer of an adenylyl group from ATP to 4'-phosphopantetheine (Ppant), producing pyrophosphate and 3'-dephospho-CoA (dPCoA). Although the crystal structures of PPATs with various ligands have been studied, the specific contributions of residues to catalytic efficiency remain unclear. Here, we present the crystal structures of Helicobacter pylori PPAT (HpPPAT) in its apo form and complexes with Ppant and ATP. Additionally, we report the structure of the HpPPAT P8A mutant bound to dPCoA, providing the first complete occupancy structure of a PPAT complex across the hexamer. In the HpPPAT:ATP complex structure, critical active site residues Thr10, His18, Arg88, and Arg91, conserved in Escherichia coli PPAT (EcPPAT), are identified. HpPPAT utilizes Pro8, Lys42, and Arg133 for ATP binding. This differs from the binding pattern observed in other bacterial PPATs. Mutations of these residues, except for Thr10 and Lys42, resulted in a complete loss of enzymatic activity. This result highlights their critical roles. Mutating Thr10 and Lys42 to alanine reduced catalytic efficiency compared to WT HpPPAT but retained substantial activity. These residues are expected to orient the nucleophile for an in-line displacement mechanism. Based on structural studies and mutagenesis data with kinetic measurements and insights from other bacterial PPATs, we propose a refined catalytic mechanism for HpPPAT that emphasizes species-specific active-site interactions. This mechanism provides a foundation structure-based drugs H. pylori infections.