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

• Published in: Biochimica et biophysica acta Vol. 1850; no. 2; pp. 393 - 400
• Main Authors: Eberle, Raphael J., Coronado, Monika A., Caruso, Icaro P., Lopes, Débora O., Miyoshi, Anderson, Azevedo, Vasco, Arni, Raghuvir K.
• Format: Journal Article
• Language: English
• Published: Netherlands 01.02.2015
• Subjects: adenine; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; caseous lymphadenitis; Circular Dichroism; copper sulfate; Copper Sulfate - chemistry; Corynebacterium pseudotuberculosis; Corynebacterium pseudotuberculosis - enzymology; Corynebacterium pseudotuberculosis - genetics; death; differential scanning calorimetry; DNA; DNA Glycosylases - chemistry; DNA Glycosylases - genetics; EDTA (chelating agent); Enzyme Stability; Escherichia coli; fluorescence emission spectroscopy; Gram-positive bacteria; Hydrogen-Ion Concentration; Iron-Sulfur Proteins - chemistry; Iron-Sulfur Proteins - genetics; livestock; mutation; pathogens; Protein Structure, Secondary; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; thermal stability; Secondary and tertiary structure; MutY; C. pseudotuberculosis; [4Fe–4S](2+) cluster; DNA repair; Spectroscopic method
• ISSN: 0304-4165; 0006-3002
• DOI: 10.1016/j.bbagen.2014.11.014

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 pH3.0 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.