Kinetic Characterization and Phosphoregulation of the Francisella tularensis 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (MEP Synthase)

Abstract

Deliberate and natural outbreaks of infectious disease underscore the necessity of effective vaccines and antimicrobial/antiviral therapeutics. The prevalence of antibiotic resistant strains and the ease by which antibiotic resistant bacteria can be intentionally engineered further highlights the need for continued development of novel antibiotics against new bacterial targets. Isoprenes are a class of molecules fundamentally involved in a variety of crucial biological functions. Mammalian cells utilize the mevalonic acid pathway for isoprene biosynthesis, whereas many bacteria utilize the methylerythritol phosphate (MEP) pathway, making the latter an attractive target for antibiotic development. In this report we describe the cloning and characterization of Francisella tularensis MEP synthase, a MEP pathway enzyme and potential target for antibiotic development. In vitro growth-inhibition assays using fosmidomycin, an inhibitor of MEP synthase, illustrates the effectiveness of MEP pathway inhibition with F. tularensis. To facilitate drug development, F. tularensis MEP synthase was cloned, expressed, purified, and characterized. Enzyme assays produced apparent kinetic constants (KMDXP = 104 µM, KMNADPH = 13 µM, kcatDXP = 2 s−1, kcatNADPH = 1.3 s−1), an IC50 for fosmidomycin of 247 nM, and a Ki for fosmidomycin of 99 nM. The enzyme exhibits a preference for Mg+2 as a divalent cation. Titanium dioxide chromatography-tandem mass spectrometry identified Ser177 as a site of phosphorylation. S177D and S177E site-directed mutants are inactive, suggesting a mechanism for post-translational control of metabolic flux through the F. tularensis MEP pathway. Overall, our study suggests that MEP synthase is an excellent target for the development of novel antibiotics against F. tularensis.

Isoprenoid biosynthesis in bacterial pathogens

“…These initial results prompted additional experiments using a more focused set of compounds to probe the IspF cytidine and Zn 2+ binding sites, and yielded a ligand with low micromolar binding affinity. An additional study by Jawaid et al (2009) examined the IspF enzyme of F. tularensis. The enzyme was cloned and expressed in E. coli, the recombinant protein was then purified and enzymically characterized, and subsequent analysis showed that IspF is an excellent target for antibiotic development.…”

Alteration of the Flexible Loop in 1-Deoxy-d-xylulose-5-phosphate Reductoisomerase Boosts Enthalpy-Driven Inhibition by Fosmidomycin

“…Fosmidomycin is a natural product produced by Streptomyces bacteria that inhibits DXR with K i values in the range of 10 –8 to 10 –7 M. 6−9 Although it is effective in the treatment of malaria caused by P. falciparum , 10,11 fosmidomycin, its acetyl analogue, and their phosphonate ester prodrugs are poorly active against M. tuberculosis . 12,13 Understanding the nature of interactions between fosmidomycin and DXR for the purposes of designing improved alternatives is therefore invaluable.…”

Current development in isoprenoid precursor biosynthesis and regulation

“…Analysis of DXR from Francisella tularensis suggests that Ser177 (equivalent to Ser185 of E. coli DXR) is phosphorylated [27]. In view of the close proximity of Ser177 to the substrate binding site, phosphorylation of Ser177 could be related to DXR activity regulation.…”