Studies on IspH: Cluster Insertion, Inhibitor Discovery, and the Role of Paramagnetic Species Detected in the E126Q Variant

Abstract

Isoprenoids or terpenoids form the largest group of natural products. They have vital biological functions in all living organisms. Despite their diversity, all isoprenoids are synthesized in all organisms from two precursors: Isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). There are however two separate pathways that lead to the synthesis of these two: The mevalonic acid (MVA) and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathways. Humans and animals use the MVA pathway while green algae, apicomplexan protozoa including Plasmodium falciparum and a large group of eubacteria such as Mycobacterium tuberculosis use the MEP pathway. The MEP pathway is an attractive target for development of anti-infective agents especially for the emerging drug resisting strains of microbes that rely solely on this pathway. The last enzyme of the MEP pathway; IspH has an oxygen sensitive iron sulfur cluster that is involved in catalysis. Recombinant IspH purifies with sub-stoichiometric cluster content that needs reconstitution before downstream applications. Contrary to the in vitro iron sulfur (Fe-S) cluster reconstitution that introduces free-iron impurities, in vivo cluster biogenesis involves complex machinery and produces a more homogeneous recombinant-protein product. Under normal conditions, E. coli uses ISC proteins for all Fe-S synthesis including IspH. Furthermore, the last step of IspH might require an A-type carrier protein; ErpA which would be involved in the delivery of de novo made [4Fe-4S]. The first topic presented here is the investigation of the role of the ISC proteins and ErpA in the cluster insertion process. The results indicate that the ISC proteins and ErpA are both required to mature IspH. The mechanism utilized by ErpA to transiently bind Fe-S cluster and later deliver it is not fully understood. ErpA has three highly conserved cysteines that can ligate one or more clusters. The protein probably involves oligomerization/de-oligomerization to be able to bind different types of Fe-S clusters and release them to proper clients. One driving force of the cluster transfer is the affinity differences between the cluster-carrier and the cluster-receiver proteins. However, evaluating cluster transfer in real time with spectroscopic tools currently in hand is a perennial challenge because it is difficult to differentiate between highly similar protein environments. Knowledge of the indirect involvement of ErpA in isoprenoids biosynthesis is of great benefit as ErpA can be an additional target for inhibitor development against isoprenoids synthesis in many pathogenic organisms. The second topic is a collaborative effort between three groups. The group of Dr. Orlando Acevedo performed in silico screening and docking of small molecules that could bind in the IspH active site. These compounds resemble the pseudo-cyclic conformation that the substrate takes in the active site of IspH. Following this, the group of Dr. Bradley Merner synthesized a series of eight carbohydrate-based bicyclo[3.3.0]-γ-octalactones, and our group evaluated the compounds by Electron Paramagnetic Resonance (EPR) spectroscopy and enzyme kinetic assays. Although all the compounds synthesized were observed to fit within the IspH active site by computation evaluation, none of them was observed to interact with the Fe-S cluster when monitored by EPR. Kinetic studies, however, showed inhibitory activity of all tested compounds against IspH. The findings from this study will be beneficial in improving the design of future compounds of the same class. The third topic revolves around the confirmation of the IspH reaction mechanism. The widely accepted IspH mechanism is based on the properties of an EPR species found in a mutant (IspHE126Q)-based intermediate that might not be catalytically relevant. IspHE126Q is kinetically inactive. Our EPR freeze quench studies suggested the EPR species from this mutant is not transient in nature since it develops slowly and does not disappear (for the longest time tested). One spectroscopic approach which is expected to provide definitive information on this question is Mӧssbauer spectroscopy studies in collaboration with Dr. Sebastian Stoan. To that end, we have paved the way toward future studies by establishing the protocol on how to label IspHE126Q enzyme with 57Fe. However, protein expression optimization is still needed to produce a protein sample with a higher cluster content that will yield a strong EPR signal and consequently will yield well resolved Mӧssbauer spectra. The last topic presented in this dissertation is another collaborative effort with the group of Dr. Daniel Lessner from the University of Arkansas. By using Methanosarcina acetivorans as a methanogen model, nitrogenase expressed under nitrogen fixing conditions with various exogenic sulfur sources was purified and the M-cluster was characterized using EPR spectroscopy. This study showed an atypical EPR signal, but no effect of the sulfur source. This dissertation is comprised of 8 chapters. Chapter 1 is a broad background and literature review on iron sulfur metalloclusters and isoprenoids biosynthesis. A recap of what is already known (or done in our lab) about IspH and ErpA together with a research statement are also included in this chapter. Chapters 2 through 6 entail actual studies that I conducted. Each chapter has its own result, discussion and conclusion sections. Chapter 7 entails general conclusions and future perspectives. All the materials and methods used in this research are listed in chapter 8. Additional detailed procedures on how we made a high salt (HS) media and the synthesis of HMBPP are in the appendix.