Mechanism of RNase cleavage by VapC from Pyrobaculum aerophilum
Duyvestyn, J. M. (2012). Mechanism of RNase cleavage by VapC from Pyrobaculum aerophilum (Thesis, Master of Science (MSc)). University of Waikato, Hamilton, New Zealand. Retrieved from http://hdl.handle.net/10289/6474
Permanent Research Commons link: http://hdl.handle.net/10289/6474
Proteins belonging to the PIN-domain family are widespread across bacteria, archaea and eukaryotes. PIN-domain structures are well characterised and contain three highly conserved acidic residues that are orientated to form the active site of the enzyme. VapC is the toxic component of a toxin-antitoxin (TA) complex, and contains a PIN-domain. The VapBC TAs are the largest TA family, and are found in expanded copy numbers in a variety of unrelated organism including the human pathogen Mycobacterium tuberculosis. It is proposed that TAs play a role in metabolic regulation, under conditions of stress. VapCs are metal dependent endoribonucleases that target specific sequences in single stranded RNA and inhibit translation by degrading mRNA transcripts. The mechanism by which VapC cleaves RNA and achieves this specificity is unknown. In this thesis, VapC 2754 from Pyrobaculum aerophilum (VapCPAE2754) is analysed, and a mechanism for catalysis of RNA cleavage by PIN-domains is proposed. Mutations were made to conserved residues in the putative active site to test the hypothesized mechanism. A fluorometric kinetic assay was designed to allow differences in activity to be accurately determined, so that conclusions about the effect of the mutations could be drawn. Mutations made to the conserved acidic amino acids eliminated RNase activity, confirming that these residues are essential to catalysis. A structure for the wild-type VapCPAE2754 was determined, containing cadmium ions and an acetate bound into the active site. Knowledge of the enzymatic mechanism of PIN domains will enable a greater understanding of this important protein family, and serve as the basis for future drug design by transition state analogue inhibiters. This is especially important in VapC enzymes due to their proposed role in the pathology of M. tuberculosis.
University of Waikato
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