Genetic Essentiality, Biochemical and Structural Properties of Fructose 1,6-bisphosphatases II

The genetic essentiality of the glpX gene in Mycobacterium tuberculosis (Mtb) was investigated by generating an unmarked deletion mutant. This mutant was characterized by its phenotype, including growth on selective media, and its in vivo survival profile in a mouse model. The glpX gene was required for eugonic growth on gluconeogenic substrates such as glycerol, acetate, and oleic acid. Mtb lacking the glpX gene not only failed to maintain the initial inoculum density(100 counts), but also failed to replicate and survive in mouse lungs (3 log difference in bacterial count compared to the wild type strain of Mtb) during the acute phase of infection. The absence of glpX in the chronic phase of infection resulted in significant mycobacterial clearance. The glpX gene encoded a functional fructose 1,6–bisphosphatase which was successfully purified using affinity capture and size exclusion chromatography. Biochemical characterization, including determination of optimal conditions for enzymatic activity was performed. Mtb FBPase belongs to the super family of lithium sensitive phosphatases which require bivalent metal ions for enzymatic activity. The crystal structures of FBPase and its complex with catalytic product fructose 6-phosphate, were solved by a standard molecular replacement method. Mtb FBPase II exists as a functional tetramer with no allosteric regulatory mechanism as compared to the classical FBPase I present in mammals and several other bacteria. The active site of Mtb FBPase is highly conserved and 100% similar to other known FBPase II enzymes, indicative of a common catalytic mechanism. The FBPase activity of the glpX-encoded protein in Francisella tularensis was confirmed. Furthermore, the protein was purified to homogeneity and crystallized following similar methods used for Mtb FBPase. Two major bottlenecks (i.e. purification and crystallization of the protein target) in the process of structure based drug discovery (SBDD) for an already validated target were overcome. The interdisciplinary studies performed herein validate FBPase II as a drug target in pathogenic bacteria and provide critical information for SBDD.