Antimicrobial resistance poses a significant global health challenge, with the World Health Organization ranking it among the top 10 global threats to public health worldwide. Hence, the urgent need for new anti-infectives targeting novel pathways.
In this context, the Methylerythritol phosphate (MEP) pathway becomes particularly significant. The MEP pathway is composed of seven enzymes and is responsible for the biosynthesis of the two isoprenoids precursors : isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). This pathway is completely absent in humans but is essential to pathogenic microorganisms such as Mycobacterium tuberculosis (Mt), Plasmodium falciparum (Pf), Pseudomonas aeruginosa (Pa) .
The aims of this project were to elucidate the crystallographic structures of the last two enzymes of the MEP pathway (IspG and IspH) from three different pathogens (P. aeruginosa, M. tuberculosis and P. falciparum) and by combining X-ray fragment screening and in silico virtual screening of chemical libraries, to identify or design new specific inhibitors of these enzymes. Due to the presence of a 4Fe-4S cluster in their active sites, all these targets are oxygen-sensitive enzymes that must be handled under anaerobic conditions using a glove-box. The first part of this work consisted in establishing a protocol for the production and purification of each of the six proteins in quantity suitable for crystallization studies. Pure proteins were obtained for all the IspH orthologues and for Pa and MtIspG.

The reactions catalyzed by IspG and IspH are dependent on an electron donating system. For Plasmodium, this system is composed of two proteins : the ferredoxin (a 2Fe-2S cluster, PfFd) and the ferredoxin-reductase (a FAD dependent protein, PfFNR). Obtaining these two active proteins in sufficient quantities was necessary for using the natural reduction system for measuring the enzymatic activities of PfIspG and PfIspH. That’s why the optimization of the expression and purification of PfFd and PfFNR was also achieved during the first part of this project. The activity tests were performed successfully with PfIspH by the group of Dr. Myriam Seemann, our close collaborators at the University of Strasbourg. They also used the Plasmodium system to screen different series of chemical compounds in vitro.
Crystallization screening under anaerobic conditions were performed for all the IspG and IspH proteins obtained. Several crystals were obtained and analyzed at the European Synchrotron Radiation Facility (ESRF). The collected data led to the elucidation of the first crystallographic structures of PaIspH and MtIspH at 1.5 Å and 2.8 Å resolution, respectively. Both enzymes show a three-leaf clover folding typical of the IspH family. PaIspH contains an incomplete 3Fe-4S cluster and a molecule of bicine coming from the crystallization buffer in its active site. The bicine is interacting with key residues involved in the substrate binding, but has no inhibitory activity. However, this has provided us structural information on possible interactions within the active site, which may help in the design of new inhibitors. For MtIspH, a shift in the third domain leads to a previously unseen opening of the active site. Surprisingly, despite this opening, MtIspH displays a 4Fe-4S cluster. This can be explained by the fact that the displacement of the third domain has brought the cluster closer to the threonine 189, which can bind and stabilize the apical iron.
​ Some of the compounds identified in silico as well as new inhibitors selected in vitro by our collaborators in Strasbourg have been used for co-crystallization assays. The new structural information combined with the in silico docking analysis and the enzymatic activity assays, opens the door for new biophysical and structural studies of these fascinating enzymes that are targeted for the development of new anti-infectives.
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