STRUCTURAL STUDIES OF SHIKIMATE AND FATTY ACID BIOSYNTHESIS PATHWAY’S ENZYMES

Abstract

Infectious diseases are the leading cause of death all over the world, with substantial contributions from microbial infections to this high rate of mortality. The traditional approaches to control bacterial infections were identified, and primarily derived from other microorganisms. These approaches mainly depend upon the disruption of the microbial growth cycle by inhibiting the synthesis, and association of the essential components of bacterial processes like cell wall synthesis, DNA replication, and protein synthesis. Although these methods and compounds were highly effective, but they have created substantial stress on target microorganism, and subsequently during the course of evolution, these bacterial pathogens rapidly selected for resistant subpopulations. The emergence of drug resistance in bacterial pathogens is pressing concerns in health sector all over the world. Antibiotic resistance in microbes is the outcome of innate genetics and physiology, which are vertically transmitted through species, and the outstanding ability of bacteria to perform the horizontal transfer of genetic material across species and genera. Since the bacterial pathogens have developed the resistance against the antibiotics, which targeted the essential pathways like cell wall synthesis, DNA (or RNA) replication, protein synthesis, and folate biosynthesis pathways it has become more important to discover antibacterial agents targeting other vital pathways or processes of bacteria. Other important pathways of bacteria that have been validated for drug development are shikimate pathway, fatty acid biosynthesis, lipid polysaccharide synthesis, cell division, two-component regulatory system, and bacterial efflux pumps. The current study focuses on the two such essential pathways, including shikimate pathway and fatty acid biosynthesis pathway. The thesis encompasses the biochemical, biophysical and structural characterization of three proteins, including chorismate mutase like domain of DAHPS from Bacillus subtilis, and β-hydroxyacylacyl carrier protein dehydratase and Malonyl-CoA: acyl carrier protein transacylase from drug-resistant pathogen Moraxella catarrhalis. More importantly, new inhibitors against these three proteins have been discovered and characterized using biochemical, biophysical, and in silico based approaches. II Shikimate pathway is responsible for the synthesis of chorismate that serves as the substrate for aromatic amino acids, folate and ubiquinone biosynthesis pathways. The first enzyme of shikimate pathway, 3-deoxy-D-arabino-heptulosonate-7-phosphatesynthase (DAHPS) catalyzes the aldol-condensation of D-erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP) to form 3-deoxy-D-arabino-heptulosonate-7- phosphate (DAHP). In this study, we have determined the crystal structure of chorismate mutase type II in complex with chlorogenic acid from Bacillus subtilis (BsCM_2) at 1.8 Å resolution. The structure provides the insight into the mode of binding of the inhibitor with enzyme and active site plasticity via the flexibility of active site loop L1'. Molecular dynamic simulation results showed that helix H2' undergoes uncoiling at the first turn and increases the mobility of loop L1'. Residues Arg45, Phe46, Arg52 and Lys76 show flexibility in side chain orientation, which may play an important role in DAHPS activity regulation by the formation of domaindomain interface. Biochemical characterization has shown that this domain has residual activity with kcat of 0.78 S-1 which is much lesser that the other reported chorismate mutases or AroH classes of CMs. Chlorogenic acid has shown the competitive mode of inhibition for BsCM_2 enzyme with Ki of 0.34 ± 0.07 mM. Additionally, the binding study showed that chlorogenic acid binds to BsCM_2 with more affinity than chorismate. The chlorogenic acid’s minimum inhibitory concentration against B. subtilis was calculated to 30 ± 5μg/ml. In the era of prevalent antibiotic resistance among pathogenic bacteria, these findings may lead to explore the possibility of validating the chlorogenic acid as an inhibitor of chorismate mutase, type II enzyme. Fatty acid biosynthesis is an essential pathway in the metabolism of microbes and most under-exploited for the development of antibacterial agents. In this study, we have characterized two important drug targets; β-hydroxyacyl-acyl carrier protein dehydratase (FabZ) and Malonyl-CoA:acyl carrier protein transacylase (FabD) from Type II FAS pathway. FabZ is an essential component of type II fatty acid biosynthesis and performs the dehydration of β-hydroxyacyl-ACP to trans-2-acyl- ACP in the elongation cycle of the FAS II pathway. FabZ is ubiquitously expressed and has uniform distribution, which makes FabZ an excellent target for developing novel drugs against pathogenic bacteria. We focused on the biochemical and biophysical characterization of FabZ from drug-resistant pathogen Moraxella III catarrhalis (McFabZ). More importantly, we have identified and characterized new inhibitors against McFabZ using biochemical, biophysical and in silico based studies. We have identified three isoflavones (daidzein, biochanin A and genistein) as novel inhibitors against McFabZ. Mode of inhibition of these compounds is competitive with IC50 values lie in the range of 6.85 μΜ to 27.7 μΜ. Conformational changes observed in the secondary and tertiary structure marked by a decrease in the helical and the sheet content in McFabZ structure upon inhibitors binding. In addition, thermodynamic data suggest that biochanin A has a strong binding affinity for McFabZ as compared to daidzein and genistein. Molecular docking studies have revealed that these inhibitors are interacting with the active site of McFabZ and making contacts with catalytic and substrate binding tunnel residues. These biochemical and biophysical findings lead to the identification of chemical scaffolds, which can lead to broad-spectrum antimicrobial drugs targeted against FabZ, and modification to existing FabZ inhibitors to improve affinity and potency. The next part of the study will focus on Malonyl-CoA:acyl carrier protein transacylase (FabD), which is an attractive target for developing broad-spectrum antibiotics. It performs initiation reaction to form malonyl-ACP, which is a key building block in fatty acid biosynthesis. In this study, we have characterized the FabD from drugresistant pathogen Moraxella catarrhalis (McFabD). More importantly, we have shown the kinetic inhibition and binding of McFabD with three new compounds from the class of aporphine alkaloids. ITC based binding studies have shown that apomorphine is binding to McFabD with a stronger affinity (KD = 4.87 μΜ) as compared to boldine (KD = 7.19 μΜ) and magnoflorine (KD = 11.7 μΜ). The possible mechanism of fluorescence quenching is found to be static with Kq values higher than 1010, which was associated with the ground state complex formation of aporphine alkaloids with McFabD. Conformational changes observed in the secondary and tertiary structure marked by the loss of helical content during the course of interactions. Molecular docking based studies have predicted the binding mode of aporphine alkaloids and it is found that these compounds are interacting in a similar fashion as known inhibitor corytuberine is interacting with McFabD. The analysis of docking poses has revealed that His 210, Leu102, Gln19, Ser101 and Arg126 are critical residues which may play important role in binding of these alkaloids to McFabD. The growth inhibition assay has shown that apomorphine has better MIC value (4-8 μg/ml) against Moraxella catarrhalis as compared to boldine and IV magnoflorine. Therefore, this study suggests that aporphine alkaloids can act as antibacterial agents and possible target of these compounds could be FabD enzyme from the FAS II pathway, and apomorphine scaffold will be more suitable among these compounds for the development of antibacterial agents. In the last part of the study, we tried to develop the multi-targeted inhibitor of FAS II pathway. In this study, we reported the 1,4-Naphthoquinone as dual inhibitors of two enzymes, FabD and FabZ of Moraxella catarrhalis. The biochemical inhibition assay has shown that NPQ has inhibited both FabZ and FabD with IC50 of 26.67 μΜ and 23.18 μΜ respectively. Mode of inhibition of NPQ against both enzymes was found to be non-competitive inhibition. Conformational changes marked by the loss of helical content were observed using CD based studies. Fluorescence quenching based binding studies have shown that NPQ has ≈7x more binding affinity for FabZ as compared to FabD enzyme which is in agreement with binding affinities determined using ITC based assay. In both cases, binding reactions were exothermic in nature driven by ethlapy change (ΔΗ<0). Molecular docking has predicted the binding poses of NPQ, and found that NPQ is interacting with active site key residues of both proteins. Additionally, we have checked the inhibitory action of aporphine alkaloids on FabZ, and BLD has inhibited FabZ with IC50 of 20.56 μΜ as compared to MNG (IC50 = 41.88 μΜ) and AMF (IC50 = 76.56 μΜ). Therefore, this study suggests that single inhibitor can be developed to target these key enzymes of FAS II pathway