Biochemical and Biophysical Characterization of Exoribonucleases from the Human Pathogen Mycobacterium tuberculosis

Mycobacterium tuberculosis remains the leading cause of mortality from a single infectious organism, infecting nearly one-third of the global population. The current emergence of multidrug-resistant strains represents a serious health problem nowadays. Moreover, regulation of gene expression through RNA metabolism is a key mechanism for bacterial growth, division and rapid accommodation to environmental conditions. Ribonucleases are enzymes present in all living organisms that play an important role in RNA processing and degradation. Particularly, ribonucleases belonging to the RNB-family are often essential for viability of prokaryotes and are implicated in the establishment of virulence of several pathogens. In the present study we aim to structurally and functionally characterize two putative exoribonucleases from the RNB-family of enzymes inM. tuberculosis. Overexpression and purification of the proteins was performed and further in vitro activity, binding and helicase assays using synthetic RNA substrates were accomplished. In parallel, a biophysical characterization proceeded with several crystallization trials and protein stability tests. We have demonstrated that both RNases are 3’-5’ exoribonucleases with different degradation properties and unravelled the importance of highly conserved residues for catalysis. Moreover, we were able to identify improved buffer formulations that increase protein stability, possibly enhancing their propensity to crystallize. The information regarding RNA metabolism in M. tuberculosis is limited and RNB-family enzymes have not been previously characterized in this important human pathogen. Thus, a complete knowledge of these ribonucleases is an approach to recognize their influence in M. tuberculosis metabolism and to better understand the post-transcriptional control in this pathogen.