|In this thesis, two projects were carried in the context of understanding the reaction mechanism of 4-Hydroxy-3-methylbut-2-enyl diphosphate reductase (also called HMBPP reductase or IspH).
The first project is the optimization of the IspH cluster content. It was found that the enzyme activity has a linear relationship with the [4Fe-4S] cluster content. In this thesis, IspH from 3 different organisms: Aquifex aeolicus, Plasmodium falciparum and Escherichia coli were co-expressed with either the isc genes or the erpA gene. In the case of the Plasmodium enzyme a higher cluster content was observed under both conditions while the Aquifex enzyme only showed improvement with the co-expression of the ErpA protein. In the case of the Escherichia enzyme the highest cluster content (70%) was found when IspH was expressed all by itself. It is not clear why the co-expression did not work properly.
The second project involved the study of the Escherichia and Aquifex enzymes under pre- and steady state conditions using an EPR-detected freeze-quench method. Both reaction intermediates and possible product signals could be detected. However, the assignment is not straightforward since different electron donors were used in the standard colorimetric assays to obtain the kinetic parameters and the freeze-quench studies to study the formation of paramagnetic reaction intermediates. Future mass spectrometry studies are needed to confirm the assignment of the observed species as reaction intermediates or product-induced signals.