Mechanistic, enzymatic and structural insights into M. tuberculosis alkyl-hydroperoxide reductase subunit C, a key enzyme of the mycobacterial antioxidant defense system as well as its interaction with its reducing partner, Thioredoxin C

• Main Author: Wong, Chui Fann
• Other Authors: Gerhard Gruber
• Format: Theses and Dissertations
• Language: English
• Published: 2018
• Subjects: DRNTU::Science::Biological sciences
• Online Access: http://hdl.handle.net/10356/73476
• Institution: Nanyang Technological University

In 2015, the World Health Organization estimated that Tuberculosis (TB) infection affects a third of the world's population and is the leading cause of mortality caused by a single infectious agent. Through the years, effective drugs were designed to target its causative agent, Mycobacterium tuberculosis (M tuberculosis). Despite this, the evolvement of resistant strains is still a cause of concern. To persist in the harsh conditions of a host's macrophage, M tuberculosis has evolved various mutations, which includes the overexpression of an alkyl-hydroperoxide reductase subunit C (MtAhpC) protein. However, few studies have been performed on the protein, MtAhpC in relation to TB infection. In the current study, recombinant Mycobacterium bovis (M bovis) (BCG Strain) AhpC (MbAhpC), which shares an identical protein sequence as MtAhpC, was generated to elucidate the structure of MbAhpC in solution. 2D projections were then performed, thereby confirming existing postulations on the dodecameric ring of AhpC in solution. With that, further characterization of MbAhpC through kinetics assay provide insights into the enzymatic k:irletics of MbAhpC. For the first time, the accurate kinetic parameters of MbAhpC, such as the catalytic efficiency (kcatl Km) and Michaelis constant (Km) were computed from the experimental data obtained. In addition, NMR titration assays revealed the reducing partner of AhpC as well as its interacting residues. Point mutations were performed on the unique N-terminus of MbAhpC. Downstream experiments like size exclusion chromatography and dynamic light scattering further highlighted the uniqueness of the conserved residues lying in the N-' terminus in maintaining the redox-oligomerization. With molecular docking and structural studies, the importance of the N-terminus of M tuberculosis was further elaborated from a structural point of view. All in all, the results presented in the current studies revealed biochemical, biophysical and structural insights into MtAhpC.