Abstract
Phosphopantetheine adenylyltransferase (PPAT) is an important enzyme that catalyzes the coenzyme A (CoA) biosynthesis in bacteria, thus it is a potential antibacterial target for drug development. To probe the critical residues governing the protein structure and ligand binding, random mutagenesis in Helicobacter pylori PPAT was performed. Here we report a unique H. pylori PPAT variant and its crystal structure in apo form. Interestingly, we found that, unlike other PPATs which are hexamers, the mutant is a domain-swapped tetramer. Generally, mutations in the hinge loop region that connect two exchanging subunits are known to induce domain swapping. However, in our study, these two mutant sites are neither located in the hinge loop region, nor make any close contacts with this loop. However, they trigger a large conformational change in the C-terminal region. The protomer structure of this mutant is in open conformation in which the C-terminal one-third of residues are intertwined with that of other polypeptide chain. Our results demonstrate that target residue when mutated to specific amino acid results in a large structural deviation in helix , altering its flexibility and thereby its ability to form a 3D domain swap. Despite structural differences, the wild-type and mutant PPAT share similar ligand binding abilities. ATP binding to these two proteins is enthalpically driven, which is dissimilar to E. coli PPAT. Finally, our unfolding studies suggest that the structural packing may affect the thermal denaturation transition for PPAT.