Structural and functional characterization of FabG4 from Mycolicibacterium smegmatis

Area of Science:

• Biochemistry
• Microbiology
• Drug Discovery

Background:

• Antimicrobial resistance is a global health crisis, with multi-drug-resistant tuberculosis (TB) posing a significant threat.
• Bacterial fatty acid synthesis (FAS) pathways differ from animal pathways, making bacterial enzymes like FabG attractive drug targets.
• High-molecular-weight FabG (HMwFabG) enzymes, such as FabG4 found in Mycobacterium tuberculosis, represent a distinct class compared to canonical low-molecular-weight FabG.

Purpose of the Study:

• To characterize the structure and function of Mycolicibacterium smegmatis FabG4 (MsFabG4), a representative HMwFabG.
• To investigate the substrate preference and cofactor usage of MsFabG4.
• To provide insights that could aid in the development of novel anti-TB drugs targeting essential bacterial enzymes.

Main Methods:

• X-ray crystallography was used to determine the structures of MsFabG4 in complex with NAD+ and in its apo form.
• Enzyme kinetics were performed to analyze the catalytic activity and substrate specificity of MsFabG4.
• Comparative analysis with other HMwFabGs and canonical FabGs was conducted.

Main Results:

• The crystal structures revealed the molecular architecture of MsFabG4.
• Kinetic analyses demonstrated that MsFabG4 preferentially utilizes NADH as a cofactor when reducing CoA substrates.
• MsFabG4 exhibits distinct biochemical properties compared to archetypal low-molecular-weight FabG enzymes.

Conclusions:

• MsFabG4 is a functional HMwFabG that preferentially uses NADH.
• Structural and functional data on MsFabG4 contribute to understanding the diversity of bacterial FabG enzymes.
• This research supports the potential of HMwFabGs as targets for developing new antimicrobial therapies, particularly against TB.