Structural and functional insights into corrinoid iron-sulfur protein from human pathogen Clostridium difficile

Area of Science:

• Biochemistry
• Molecular Biology
• Microbiology

Background:

• Clostridium difficile infection (CDI) is a significant healthcare-associated infection.
• The Wood-Ljungdahl pathway is crucial for the survival of C. difficile and absent in humans.
• Key enzymes in this pathway are potential therapeutic targets for CDI treatment.

Purpose of the Study:

• To clone, express, and characterize the Corrinoid iron-sulfur protein (CoFeSP) from Clostridium difficile (CoFeSP_Cd) for the first time.
• To investigate the structure and function of CoFeSP_Cd.
• To provide a molecular basis for developing novel anti-CDI therapeutics.

Main Methods:

• Homology modeling, spectroscopy, electrochemistry, kinetic studies, and molecular docking were employed.
• Characterization of CoFeSP_Cd's metal centers (corrinoid cofactor and [4Fe-4S] cluster) using metal analysis, structural modeling, UV-Vis, EPR, and electrochemistry.
• Determination of methyl transfer activity using kinetic studies involving methyl transferase (MeTr_Cd) and CH₃-H₄folate (CH₃-THF).

Main Results:

• CoFeSP_Cd was successfully cloned, expressed in E. coli, and characterized.
• The study elucidated the structural and functional properties of CoFeSP_Cd, including its two metal centers.
• Methyl transfer activity of CoFeSP_Cd was quantified, providing insights into its role in the Wood-Ljungdahl pathway.

Conclusions:

• The characterization of CoFeSP_Cd provides crucial molecular insights into its function.
• This research lays the foundation for designing targeted drugs against C. difficile.
• Understanding CoFeSP_Cd is vital for developing innovative strategies to combat CDI.