ATP-specificity of succinyl-CoA synthetase from Blastocystis hominis.

Succinyl-CoA synthetase (SCS) catalyzes the only step of the tricarboxylic acid cycle that leads to substrate-level phosphorylation. Some forms of SCS are specific for ADP/ATP or for GDP/GTP, while others can bind all of these nucleotides, generally with different affinities. The theory of `gatekeeper' residues has been proposed to explain the nucleotide-specificity. Gatekeeper residues lie outside the binding site and create specific electrostatic interactions with incoming nucleotides to determine whether the nucleotides can enter the binding site. To test this theory, the crystal structure of the nucleotide-binding domain in complex with Mg2+-ADP was determined, as well as the structures of four proteins with single mutations, K46ßE, K114ßD, V113ßL and L227ßF, and one with two mutations, K46ßE/K114ßD. The crystal structures show that the enzyme is specific for ADP/ATP because of interactions between the nucleotide and the binding site. Nucleotide-specificity is provided by hydrogen-bonding interactions between the adenine base and Gln20ß, Gly111ß and Val113ß. The O atom of the side chain of Gln20ß interacts with N6 of ADP, while the side-chain N atom interacts with the carbonyl O atom of Gly111ß. It is the different conformations of the backbone at Gln20ß, of the side chain of Gln20ß and of the linker that make the enzyme ATP-specific. This linker connects the two subdomains of the ATP-grasp fold and interacts differently with adenine and guanine bases. The mutant proteins have similar conformations, although the L227ßF mutant shows structural changes that disrupt the binding site for the magnesium ion. Although the K46ßE/K114ßD double mutant of Blastocystis hominis SCS binds GTP better than ATP according to kinetic assays, only the complex with Mg2+-ADP was obtained.

Engineering Nucleotide Specificity of Succinyl-CoA Synthetase in Blastocystis: The Emerging Role of Gatekeeper Residues.

Charged, solvent-exposed residues at the entrance to the substrate binding site (gatekeeper residues) produce electrostatic dipole interactions with approaching substrates, and control their access by a novel mechanism called "electrostatic gatekeeper effect". This proof-of-concept study demonstrates that the nucleotide specificity can be engineered by altering the electrostatic properties of the gatekeeper residues outside the binding site. Using Blastocystis succinyl-CoA synthetase (SCS, EC 6.2.1.5), we demonstrated that the gatekeeper mutant (ED) resulted in ATP-specific SCS to show high GTP specificity. Moreover, nucleotide binding site mutant (LF) had no effect on GTP specificity and remained ATP-specific. However, via combination of the gatekeeper mutant with the nucleotide binding site mutant (ED+LF), a complete reversal of nucleotide specificity was obtained with GTP, but no detectable activity was obtained with ATP. This striking result of the combined mutant (ED+LF) was due to two changes; negatively charged gatekeeper residues (ED) favored GTP access, and nucleotide binding site residues (LF) altered ATP binding, which was consistent with the hypothesis of the "electrostatic gatekeeper effect". These results were further supported by molecular modeling and simulation studies. Hence, it is imperative to extend the strategy of the gatekeeper effect in a different range of crucial enzymes (synthetases, kinases, and transferases) to engineer substrate specificity for various industrial applications and substrate-based drug design.

The potential of liposome-encapsulated ciprofloxacin as a tularemia therapy.

Liposome-encapsulation has been suggested as method to improve the efficacy of ciprofloxacin against the intracellular pathogen, Francisella tularensis. Early work with a prototype formulation, evaluated for use against the F. tularensis live vaccine strain, showed that a single dose of liposomal ciprofloxacin given by the intranasal or inhalational route could provide protection in a mouse model of pneumonic tularemia. Liposomal ciprofloxacin offered better protection than ciprofloxacin given by the same routes. Liposomal ciprofloxacin has been further developed by Aradigm Corporation for Pseudomonas aeruginosa infections in patients with cystic fibrosis and non-cystic fibrosis bronchiectasis. This advanced development formulation is safe, effective and well tolerated in human clinical trials. Further evaluation of the advanced liposomal ciprofloxacin formulation against the highly virulent F. tularensis Schu S4 strain has shown that aerosolized CFI (Ciprofloxacin encapsulated in liposomes for inhalation) provides significantly better protection than oral ciprofloxacin. Thus, liposomal ciprofloxacin is a promising treatment for tularemia and further research with the aim of enabling licensure under the animal rule is warranted.

Assessment of antimicrobial peptide LL-37 as a post-exposure therapy to protect against respiratory tularemia in mice.

Early activation of the innate immune response is important for protection against infection with Francisella tularensis live vaccine strain (LVS) in mice. The human cathelicidin antimicrobial peptide LL-37 is known to have immunomodulatory properties, and therefore exogenously administered LL-37 may be suitable as an early post-exposure therapy to protect against LVS infection. LL-37 has been evaluated for immunostimulatory activity in uninfected mice and for activity against LVS in macrophage assays and protective efficacy when administered post-challenge in a mouse model of respiratory tularemia. Increased levels of pro-inflammatory cytokine IL-6, chemokines monocyte chemoattractant protein 1 (MCP-1) and CXCL1 with increased neutrophil influx into the lungs were observed in uninfected mice after intranasal administration of LL-37. Following LVS challenge, LL-37 administration resulted in increased IL-6, IL-12 p70, IFNγ and MCP-1 production, a slowing of LVS growth in the lung, and a significant extension of mean time to death compared to control mice. However, protection was transient, with the LL-37 treated mice eventually succumbing to infection. As this short course of nasally delivered LL-37 was moderately effective at overcoming the immunosuppressive effects of LVS infection this suggests that a more sustained treatment regimen may be an effective therapy against this pathogen.