Chemical and thermal influence of the [4Fe-4S]2Â + cluster of A/G-specific adenine glycosylase from Corynebacterium pseudotuberculosis

The gram-positive bacteria Corynebacterium pseudotuberculosis, the causative agent of caseous lymphadenitis in livestock significantly reduces productivity and often causes death. The adenine/guanine-specific DNA glycosylase (MutY) prevents mutations in the DNA of the pathogen and a unique feature of the MutY protein family is the [4Fe-4S]2 + cluster that interlinks two protein subdomains. MutY from C. pseudotuberculosis was expressed in E. coli and purified, the CD experiments indicate a high content of α-helices and random coiled secondary structure and a typical near-UV CD fingerprint for the [4Fe-4S]2 + cluster. EDTA and copper sulfate possess a strong destabilizing effect on the [4Fe-4S]2 + cluster. UV-vis and fluorescence spectroscopy results demonstrate that between pH 3.0 and 4.0 the integrity of the [4Fe-4S]2 + cluster is destroyed. To investigate the thermal stability of the protein differential scanning calorimetry and fluorescence spectroscopy were used and the Tm was determined to be 45 °C. The analysis presented provides information concerning the protein stability under different physio-chemical conditions.