Structural and functional insights of biological macromolecules associated with infectious diseases single particle cryo electron microscopy based studies

Abstract

In the global context, particularly in India, the two most important infectious diseases among many others are Tuberculosis (TB), an age-old disease and COVID-19, a new-age disease. This thesis focuses on some of the key proteins responsible for their pathogenesis. In the second chapter, structural and functional characterization of Cystathionine-and#946;-synthase from Mycobacterium tuberculosis (Mtb) was done using single-particle cryo-electron microscopy. CBS is the first enzyme of the transsulfuration pathway. This pathway produces hydrogen-di-sulfide (H2S) that scavenge reactive oxygen species (ROS) and reactive nitrogen species (RNS), thereby protecting Mtb against oxidative stress. In this current study, recombinant MtbCBS protein was purified as a tetramer followed by several biochemical and biophysical studies to characterize oligomeric states, enzyme activities and allosteric activation of this enzyme. Native MtbCBS structure was resolved at a global resolution of 3.6 and#8491; resolution calculated at 0.143 Fourier Shell Correlation (FSC). The local resolution showed the resolution of the core area between 3-3.2 and#8491;, whereas the periphery area was resolved at a resolution of 3.4-3.6 and#8491;. The atomic model of native MtbCBS was determined and demonstrated the domain boundaries. It is composed of N-terminal catalytic core region, C-terminal Bateman module and a 32 amino acid long linker between catalytic core and Bateman module. The active site is lysine 44, where pyridoxal phosphate (PLP) binds as a cofactor. Two Bateman module from two different monomers interacts antiparallelly during tetramerization. Site-directed mutagenesis was performed for the amino acids (L454, R450, S390, E388 and I357), present at the tetramer interface, followed by enzyme activities of mutated MtbCBS was measured. Current studies indicate that I357A mutation modulates the partial conversion (not the entire population) of tetramer to the dimer. Interestingly the basal specific activity of I357A mutant showed two-fold higher activity than native...