These include K161, which forms a Schiff base with the first substrate to bind the enzyme, and a catalytic triad comprised of Y107, T44 and Y133, which are strongly conserved in all DHDPS enzymes characterized to date including S. pneumoniae. Given the clinical importance of S. pneumoniae and the rise in multi-drug resistance in this Gram-positive pathogen, the aims of this study were to Rapamycin mTOR inhibitor determine the phenotype of a DHDPS gene knock mutant of S., pneumoniae; characterize the solution properties, stability and catalytic activity of SpDHDPS; and determine the high-resolution crystal 
structure of the enzyme to afford structure-based drug design strategies in future studies. Solution studies also showed Sp-DHDPS possesses significantly greater thermostability compared to Ec-DHDPS. We subsequently demonstrated using analytical ultracentrifugation that the enhanced thermostability of SpDHDPS is contributed by a 45-fold tighter tetramer-dimer dissociation constant. We therefore determined the three-dimensional structure of Sp-DHDPS using X-ray crystallography to provide insight into the enhanced thermal and thermodynamic stability. The origin of the enhanced thermal stability is clearly revealed in the resolution crystal structure of Sp-DHDPS that shows a tetrameric structure of the enzyme, consistent with our solution studies. The tertiary and quaternary structure architecture of Sp-DHDPS is very similar to Ec-DHDPS with an overall RMSD for superposition of the tetramers of 1.1 ?, as well as to other structurally characterized bacterial DHDPS enzymes;. However, significant structural differences are observed between Sp-DHDPS and Ec-DHDPS at the subunit interfaces. We show that there is an increase of 60 ?2 and 300 ?2 in solvent-inaccessible Bortezomib surface area at the ‘tight’ dimer and ‘weak’ dimer interfaces of Sp-DHDPS, respectively. This significant increase in SISA, in combination with the greater proportion of hydrogen bonding residues at the ‘tight’ dimer interface, as well as the presence of residues participating in salt bridge interactions at the ‘weak’ dimer interface, is consistent with the 45-fold lower tetramer-dimer dissociation constant for Sp-DHDPS, and to its considerably higher thermal stability. Consistent with previous studies of other DHDPS enzymes, the residues that form interactions at the ‘weak’ dimer interface are poorly conserved in SpDHDPS, whereas strong conservation is observed at the ‘tight’ dimer interface where the active sites are located. Given that recent studies show dimeric mutants of DHDPS have significantly attenuated catalytic function compared to the wild-type tetramers, the poor conservation at the ‘weak’ dimer interface offers potential for the design of pathogen-specific antimicrobial agents, particularly given that protein-protein interfaces represent highly specific drug targets. Indeed, with the increase in drug-resistant bacteria linked to the overuse and misuse of broad spectrum antibiotics, exploiting the ‘weak’ dimer interface of SpDHDPS may provide a means to negate the incidence of broad spectrum drug resistance. In conclusion, through gene knock-out studies, circular dichroism spectroscopy, analytical ultracentrifugation, dynamic light scattering, enzyme kinetics and X-ray crystallography studies, we demonstrate that Sp-DHDPS is an essential, active and thermostable tetramer. Our work offers insight into rational drug design strategies targeting multiple sites of the enzyme to afford the discovery of novel antibiotic agents with potential to negate drug resistance. Ecosystems are characterized and understood from the fundamental relationships between predator and prey. When linked vertically and horizontally these relationships form food webs, which depict how energy flows through ecosystems and demonstrate how various components of the web interact.