Purification of a novel enzyme in methionine biosynthesis from tuberculosis

Abstract

This study focuses on the characterization and purification of enzymes involved in methionine biosynthesis, specifically MetC, alongside the cofactor pyridoxal phosphate (PLP), intending to understand their roles in bacterial pathogenicity and explore their potential as targets for antimicrobial drug discovery. Methionine biosynthesis is a crucial pathway in bacteria, influencing not only the growth and survival but also biofilm formation as well as virulence which makes it an attractive target for novel therapeutic agents. The study involves the expression of MetC enzymes followed by purification using affinity chromatography. Initial analyses including UV-visible spectroscopy to assess the integrity of the cofactor PLP and to determine the purity of the enzyme. Despite successful purification, kinetic assays showed no detectable activity which prompted further investigations into structural and functional integrity of the enzyme. SDS-PAGE analysis was conducted which confirmed the expected molecular weights, ruling out degradation or incomplete purification as a potential issue. Additionally, the negative staining technique was applied to allow visualization of the protein samples under electron microscopy without the need for crystallization which revealed the overall morphology and structural inactivity of the enzyme. Overall, the study seeks to elucidate the factors impacting enzyme activity which is critical for developing effective inhibitors targeting methionine biosynthesis. By understanding the structural and functional differences of the enzyme, the study hopes to contribute to the broader field of antimicrobial drug discovery, offering new avenues for the development of therapies against bacterial pathogens resistant to the current treatment options. Furthermore, the findings from this research could have significant implications for the design of novel antibiotics, particularly those that target metabolic pathways that are unique to bacteria.