Crystal Structure of Inorganic Pyrophosphatase from Acinetobacter baumannii

Crissy L. Tarver¹, Qunying Yuan², Marc Pusey², Joseph Ng²

¹ University of Alabama in Huntsville, 301 Sparkman Drive, Huntsville, AL, 35899

² iXpressGenes, Inc., 601 Genome Way, Huntsville, AL, 35806

An inorganic pyrophosphate molecule (PPi) is hydrolyzed into two inorganic phosphate (Pi) molecules by inorganic pyrophosphatase (IPPase).  IPPases are crucial for the growth and development of cells in archaea, eukaryotes, and prokaryotes because the hydrolysis of the product PPi is necessary to maintain the forward direction of metabolic reactions.  IPPases are divided into two families, family I and family II.  Family I include yeast and human IPPases, while family II is found in numerous bacterial and archaeal species.  These families reveal no sequence homology, and family II IPPases prefer Mn²⁺ over Mg²⁺ as the divalent metal ion used for catalysis.  A. baumannii is an opportunistic gram-negative bacillus that is aerobic and multidrug-resistant (MDR).  This pathogen apparently has active site residues typical of both families.  We are interested in exploring the use of IPPases as drug targets.  In order to attempt structural-based drug target design, this enzyme was cloned, expressed, purified, and characterized.  Three commercial and one in-house designed screen were used with the sitting drop vapor diffusion method to search for lead crystallization conditions.  After 6 weeks the scored results were subjected to AED analysis and the results used to generate a new 96 condition screen.  Eight lead conditions from the AED screen plate giving crystals after 1 week were selected and used to set up an optimization plate having increasing concentrations of glycerol (0, 10, 20, and 30 %) to prepare crystals for diffraction analysis.   A protein crystal was retrieved from a well containing sodium cacodylate pH 6.5, 0.9 M monoammonium phosphate, 0.2 M Na/K tartrate, and 30% glycerol for x-ray diffraction.  The crystal was retrieved on a 0.2mm loop and cryocooled in liquid nitrogen.  Diffraction data was collected using the SBCbeamline BM19 at Argonne National Lab.  The space group for the diffracted crystal was P222₁, and the structure was determined to 2.1 Å resolution.  The structure was solved by molecular replacement using E. coli IPPase as a model.