Structure and Function of N-Acetylmannosamine Kinases from Pathogenic Bacteria

Area of Science:

• Biochemistry
• Structural Biology
• Microbiology

Background:

• Pathogenic bacteria utilize sialic acids for carbon and nitrogen, with N-acetylmannosamine kinase (NanK) being crucial in this catabolism.
• The conserved zinc-finger (ZnF) motif in ROK superfamily enzymes, including NanK, is vital for structure and function.
• Fusobacterium nucleatum NanK (Fn-NanK) lacks the ZnF motif, raising questions about its enzymatic activity and structural adaptations.

Purpose of the Study:

• To investigate the functional properties of Fn-NanK lacking the ZnF motif.
• To compare the activity of Fn-NanK with ZnF-containing NanK enzymes from related pathogenic bacteria.
• To elucidate the structural basis for Fn-NanK's function in the absence of the ZnF motif.

Main Methods:

• Kinetic and thermodynamic studies were performed on Fn-NanK and its substrates (ManNAc and ATP).
• Comparative activity analysis was conducted with NanK enzymes from Haemophilus influenzae (Hi-NanK), Pasteurella multocida (Pm-NanK), and Vibrio cholerae (Vc-NanK).
• Crystal structures of Pm-NanK and Hi-NanK were solved and compared with Fn-NanK and human GNE (hMNK).

Main Results:

• Fn-NanK exhibited a 10-fold lower substrate binding affinity compared to ZnF-containing NanKs.
• Structural comparisons revealed high structural similarity between Fn-NanK and other NanK enzymes despite sequence differences.
• Hydrophobic residues in Fn-NanK substitute for the ZnF motif, forming a cluster that orients ManNAc in the active site.

Conclusions:

• ZnF-containing and ZnF-lacking NanK enzymes are functionally similar but differ in metal ion requirements.
• Structural modifications, specifically a hydrophobic cluster, compensate for the absence of the ZnF motif in Fn-NanK.
• These findings provide insights into the evolutionary adaptability of NanK enzymes in pathogenic bacteria.