W0093

Structural and Mechanistic Studies of a Pantothenate Synthetase from Mycobacterium tuberculosis. Shuishu Wang and David Eisenberg, Howard Hughes Medical Inst., UCLA-DOE Inst. of Genomics and Proteomics, Molecular Biology Inst., UCLA, Box 951570, Los Angeles, CA 90095-1570, USA.

Pantothenate biosynthesis is essential for the virulence of Mycobacterium tuberculosis, and this pathway thus presents potential drug targets against tuberculosis. Pantothenate synthetase (PS) catalyzes the last step reaction of the biosynthetic pathway, the ATP dependent condensation of pantoate and β-alanine to form pantothenate. We determined the crystal structure of the PS from M. tuberculosis, and its complexes with AMPCPP, pantoate, and a reaction intermediate, pantoyl adenylate with resolutions from 1.6 Å to 2 Å. The structure reveals a dimer, and each subunit has two domains with tight association between domains. The active site cavity is on the N-terminal domain, partially covered by the C-terminal domain. One wall of the active site cavity is flexible, which allows substrates to diffuse into the active site when crystals are soaked in solutions containing substrates. This flexible wall becomes ordered when the reaction intermediate is in the active site, thus functioning as a gate to the active site cavity. The crystal structures of the complexes with AMPCPP and pantoate indicate that the enzyme binds ATP and pantoate tightly in the active site, and brings the carboxyl oxygen of pantoate near the α-phosphorus atom of ATP for an in-line nucleophilic attack. Binding of β-alanine can occur only after pantoyl adenylate is formed inside the active site cavity. The tight binding of the intermediate pantoyl adenylate suggests that non-reactive analogs of pantoyl adenylate may be good inhibitors to the PS enzyme.