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The present work illustrates the structural and biochemical characterization of two diverse proteins, BadI and MenD from Rhodopseudomonas palustris and Staphylococcus aureus, respectively.
BadI or 2-ketocyclohexanecarboxyl-CoA is one of the key enzymes involved in the anaerobic degradation of aromatic compounds. The degradation of aromatic compounds is a vital process for the maintenance of the biogeochemical carbon cycle and bioremediation of xenobiotic compounds, which if present at higher concentrations can cause potential hazards to humans. Due to the relatively inert nature of aromatic compounds, enzymes catalyzing their degradation are of special interest for industrial applications. BadI is one of the key enzymes involved in the anaerobic degradation of aromatic compounds into an aliphatic moiety.
The major focus of this study was to provide mechanistic insights into the reaction catalyzed by BadI. BadI belongs to the crotonase superfamily and shares high sequence homology with the family members of MenB or dihydroxynaphthoate synthase. BadI is known to catalyze the cleavage of the cyclic ring of 2-ketocyclohexane carboxyl-CoA by hydrolyzing the C-C bond leading to the formation of the aliphatic compound pimelyl CoA. On the other hand MenB catalyzes the condensation reaction of o-succinylbenzoyl-CoA to dihydroxylnaphthoyl-CoA. A comprehensive amino acid sequence analysis between BadI and MenB showed that the active site residues of MenB from Mycobacterium tuberculosis (mtMenB) are conserved in BadI from Rhodopseudomonas palustris. MenB is involved in the menaquinone biosynthesis pathway and is a potential drug target against Mycobacterium tuberculosis as it has no known human homologs. Due to the high homology between MenB and BadI and the inability to obtain MenB-inhibitor complex structures we extended our interest to BadI to explore a potential substitute model for mtMenB as a drug target.
In addition, BadI possesses some unique mechanistic characteristics. As mentioned before, it hydrolyzes the substrate via a retro Dieckmann’s reaction contrasting its closest homolog MenB that catalyzes a ring closing reaction through a Dieckmann’s reaction. Nevertheless the active site residues in both enzymes seem to be highly conserved. We therefore decided to pursue the structural characterization of BadI to shed light on the similarities and differences between BadI and MenB and thereby provide some insights how they accomplish the contrasting reactions described above.
We determined the first structures of BadI, in its apo and a substrate mimic bound form. The crystal structures revealed that the overall fold of BadI is similar to other crotonase superfamily members. However, there is no indication of domain swapping in BadI as observed for MenB. The absence of domain swapping is quite remarkable because the domain swapped C-terminal helical domain in MenB provides a tyrosine that is imperative for catalysis and is also conserved in the BadI sequence. Comparison of the active sites revealed that the C-terminus of BadI folds onto its core in such a way that the conserved tyrosine is located in the same position as in MenB and can form interactions with the ligand molecule. The structure of BadI also confirms the role of a serine and an aspartate in ligand interaction, thus validating that the conserved active site triad participates in the enzymatic reaction. The structures also reveal a noteworthy movement of the active site aspartate that adopts two major conformations. Structural studies further illuminated close proximity of the active site serine to a water and chlorine molecule and to the carbon atom at which the carbonyl group of the true substrate would reside. Biochemical characterization of BadI using enzyme kinetics validated that the suggested active site residues are involved in substrate interaction. However, the role of these residues is very distinct, with the serine assuming a major role. Thus, the present work ascertain the participation of putative active site residues and demonstrates that the active site residues of BadI adopt very distinctive roles compared to their closest homolog MenB.
The MenD protein also referred to as SEPHCHC (2-succinyl-5-enolpyruvyl-6- hydroxy-3-cyclohexene-1-carboxylic acid) synthase is one of the enzymes involved in menaquinone biosynthesis in Staphylococcous aureus. Though S. aureus is usually considered as a commensal it can act as a remarkable pathogen when it crosses the epithelium, causing a wide spectrum of disorders ranging from skin infection to life threatening diseases. Small colony variants (SCVs), a slow growing, small sized subpopulation of the bacteria has been associated with persistent, recurrent and antibiotic resistant infections. These variants show autotrophy for thiamine, menaquinone or hemin. Menaquinone is an essential component in the electron transport pathway in gram-positive organisms. Therefore, enzymes partaking in this pathway are attractive drug targets against pathogens such as Mycobacterium tuberculosis and Bacillus subtilis. MenD, an enzyme catalyzing the first irreversible step in the menaquinone biosynthetic pathway has been implicated in the SCV phenotype of S. aureus. In the present work we explored biochemical and structural properties of this important enzyme.
Our structural analysis revealed that despite its low sequence identity of 28%, the overall fold of staphylococcal MenD (saMenD) is similar to Escherichia coli MenD (ecMenD) albeit with some significant disparities. Major structural differences can be observed near the active site region of the protein and are profound in the C-terminal helix and a loop near the active site. The loop contains critical residues for cofactor binding and is well ordered only in the ecMenD-ThDP structure, while in the apo and substrate bound structures of ecMenD the loop is primarily disordered. In our saMenD structure the loop is for the first time completely ordered in the apo form and displays a novel conformation of the cofactor-binding loop. The loop adopts an unusual open conformation and the conserved residues, which are responsible for cofactor binding are located too far away to form a productive complex with the cofactor in this conformation. Additionally, biochemical studies in conjugation with the structural data aided in the identification of the substrate-binding pocket and delineated residues contributing to its binding and catalysis. Thus the present work successfully divulged the unique biochemical and structural characteristics of saMenD.
The SARS virus is the etiological agent of the severe acute respiratory syndrome, a deadly disease that caused more than 700 causalities in 2003. One of its viral proteins, the SARS coronavirus main protease, is considered as a potential drug target and represents an important model system for other coronaviruses. Despite extensive knowledge about this enzyme, it still lacks an effective anti-viral drug. Furthermore, it possesses some unusual features related to its active-site region. This work gives atomistic insights into the SARS coronavirus main protease and tries to reveal mechanistic aspects that control catalysis and inhibition. Thereby, it applies state-of-the-art computational methods to develop models for this enzyme that are capable to reproduce and interpreting the experimental observations. The theoretical investigations are elaborated over four main fields that assess the accuracy of the used methods, and employ them to understand the function of the active-site region, the inhibition mechanism, and the ligand binding. The testing of different quantum chemical methods reveals that their performance depends partly on the employed model. This can be a gas phase description, a continuum solvent model, or a hybrid QM/MM approach. The latter represents the preferred method for the atomistic modeling of biochemical reactions. A benchmarking uncovers some serious problems for semi-empirical methods when applied in proton transfer reactions. To understand substrate cleavage and inhibition of SARS coronavirus main protease, proton transfer reactions between the Cys/His catalytic dyad are calculated. Results show that the switching between neutral and zwitterionic state plays a central role for both mechanisms. It is demonstrated that this electrostatic trigger is remarkably influenced by substrate binding. Whereas the occupation of the active-site by the substrate leads to a fostered zwitterion formation, the inhibitor binding does not mimic this effect for the employed example. The underlying reason is related to the coverage of the active-site by the ligand, which gives new implications for rational improvements of inhibitors. More detailed insights into reversible and irreversible inhibition are derived from in silico screenings for the class of Michael acceptors that follow a conjugated addition reaction. From the comparison of several substitution patterns it becomes obvious that different inhibitor warheads follow different mechanisms. Nevertheless, the initial formation of a zwitterionic catalytic dyad is found as a common precondition for all inhibition reactions. Finally, non-covalent inhibitor binding is investigated for the case of SARS coranavirus main protease in complex with the inhibitor TS174. A novel workflow is developed that includes an interplay between theory and experiment in terms of molecular dynamic simulation, tabu search, and X-ray structure refinement. The results show that inhibitor binding is possible for multiple poses and stereoisomers of TS174.
KasA is a key enzyme which plays an essential part in the biosynthetic pathway of mycolic acids, the building block of cell wall in Mycobacterium tuberculosis. Its importance was demonstrated by the finding that the depletion of KasA leads to the cell lysis of Mycobacterium tuberculosis. Since Mycobacterium tuberculosis is a pathogen of tuberculosis, the second leading cause of death from an infectious disease worldwide, KasA has drawn attention as one of the attractive drug targets against tuberculosis. Due to the emergence of extensively drug-resistant strains which make most of the known antibiotics for treating tuberculosis ineffective, it became an urgent issue to develop new drugs against tuberculosis. In chapter 3.1, the protonation state of the catalytic residues in the resting state was mainly addressed. The FEP computation and MD simulations were employed for this investigation, and the results showed that the zwitterionic state is most probable. To underpin this conclusion with more solid data, The PESs for the proton transfer between the neutral and zwitterionic state were computed in the context of QM/MM. However, due to the strong dependency of the QM/MM optimization on the initial structure, it was not possible to obtain consistent results from these computations. To circumvent this problem, QM/MM based umbrella sampling was carried out with a semi-empirical method (RM1), and the resulting PMF surface indicated that the zwitterionic state is more stable than the neutral state. In chapter 3.2, the protonation state of significant residues in the acyl-enzyme state was investigated. Unlike other catalytic residues, the protonation state of His311 is ambiguous in the acyl-enzyme state, and different decarboxylation mechanisms can be derived depending on the protonation state of His311 in the acyl-enzyme state. Therefore, FEP computations were carried out to find most probable protonation state of His311 in terms of free energy, and the results showed that the pKa value at Nδ is considerably lowered by the enzyme environment while that of Nε is not. Additionally, the PMF profiles for the proton transfer between Lys340 and Glu354 were computed using QM/MM based umbrellas sampling method, and the results showed that the property of the Lys340/Glu354 pair is neutral rather than ionic when His311 is protonated at Nε. Moreover, a relatively larger ionic character of the Lys340/Glu354 pair when His311 is doubly protonated provides a valuable insight into how the Lys340/Glu354 pair plays a role in shifting the protonated state from Nδ to Nε in His311 after the acyl-transfer step. Overall, the results demonstrated that His311 is neutral and protonated at Nε, and the Lys340/Glu354 pair is also neutral in the acyl-enzyme state. Those computational results lead to the conclusion that the decarboxylation reaction is facilitated by an oxyanion hole which is comprised of two catalytic histidines. In chapter 3.3, the protonation state of catalytic residues in the resting state was revisited because a recent benchmark study showed that the employed semi-empirical method (RM1) in chapter 3.1 tends to overestimate the stabilization of the zwitterionic state. Furthermore, the Lys340/Glu354 pair was considered as purely ionic in chapter 3.1, while it actually has a mixed neutral and ionic character as demonstrated in chapter 3.2. The new investigations employed a larger QM region including the Lys340/Glu354 pair with the BLYP/6-31G** approach, which was proven to be accurate enough for the present purpose by benchmark computations. The new results from the QM/MM MD and FEP computations indicated the catalytic residues to be neutral most probably in the resting state, and this in turn brought up the question how KasA can be activated to initiate the catalytic reaction. On the basis of the results from the MD simulations and FEP computations for the His311Ala mutant in chapter 3.1, we hypothesized that the open conformation of Phe404 would trigger the activation of the catalytic residues by the formation of a strong hydrogen bond. The QM/MM MD simulation proved that the activation of the catalytic residues can indeed be accomplished by the open conformation of Phe404 we suggested, and the corresponding force field based PMF profile also indicated that this conformational change is energetically feasible. The distribution of hydrophilic and hydrophobic residues in the malonyl binding pocket in conjunction with our computational results further provided a valuable insight into the detailed process how the catalytic residues is activated upon the substrate entering.