Abstract
Staphylococcus aureus is a major human pathogen responsible for a variety of clinical infections, becoming increasingly resistant to antibiotics. To address this challenge, there is a need to identify new cellular targets and innovative approaches to expand treatment options. One such target is thymidine kinase (TK), a crucial enzyme in the pyrimidine salvage pathway, which plays a key role in the phosphorylation of thymidine, an essential component in DNA synthesis and repair. In this study, we have successfully cloned, expressed, and purified the TK protein. A comprehensive investigation into how different pH levels affect the structure and functional activity of TK, using a combination of spectroscopy, classical molecular dynamics simulations, and enzyme activity assays was conducted. Our study revealed that variation in pH disrupts secondary and tertiary structures of TK with noticeable aggregate formation at pH 5.0. Enzyme activity studies demonstrated that TK exhibited its maximum kinase activity within the physiological pH range. These findings strongly suggest a connection between structural changes and enzymatic activity, which was further supported by the agreement between the spectroscopic features we measured and the results of our MD simulations. Our study provides a deeper insight into the structural features of TK, which could potentially be harnessed for the development of therapeutic strategies aimed at combatting infectious diseases. Conformational dynamics plays an essential role in the design and development of effective inhibitors. Considering the effects of pH on the conformational dynamics of TK, our findings may be implicated in the development of potent and selective inhibitors.Communicated by Ramaswamy H. Sarma.