Backbone resonance assignment and dynamics of 110 kDa hexameric inorganic pyrophosphatase from Mycobacterium...

Family I soluble inorganic pyrophosphatases (PPases; EC 3.6.1.1) are enzymes essential for all organisms. They hydrolyze inorganic pyrophosphate, thus providing the driving force for numerous biosynthetic reactions. Soluble PPases retain enzymatic activity only in multimeric forms. PPases from various organisms are extensively studied by X-ray crystallography but until now there was no information on their structure and dynamics in solution. Hexameric 110 kDa (6 × 18.3 kDa) PPase from Mycobacterium tuberculosis (Mt-PPase) is a promising target for the rational design of potential anti-tuberculosis agents. In order to use NMR techniques in functional studies of Mt-PPase and rational design of the inhibitors for this enzyme, it is necessary to have information on the backbone ¹H, ¹³C and ¹⁵N resonance assignments. Samples of Mt-PPase enriched with 99% of ¹³C and ¹⁵N isotopes, and 95% of ²H were obtained using recombinant protein expression in an isotopically-labeled medium and effective heat-shock protocol for the deuterium-to-hydrogen exchange of the amide groups. Backbone resonance assignment was achieved for more than 95% of the residues. It was found that the secondary structure of Mt-PPase in solution corresponds well to the crystal structure of this protein. Protein backbone dynamics were studied using ¹⁵N NMR relaxation experiments. Determined resonance assignments and dynamic properties provide the basis for the subsequent structure-based design of novel inhibitors of Mt-PPase-potential anti-tuberculosis drugs.