Mapping enzymatic functionalities of mannuronan C-5 epimerases and their modular units by dynamic force spectroscopy

Area of Science:

• Biochemistry
• Polymer Science
• Enzymology

Background:

• Alginates are polysaccharides with variable mannuronic acid (M) and guluronic acid (G) residues.
• AlgE enzymes catalyze the C-5 epimerization of M to G, influencing alginate structure and function.
• The specific roles of AlgE enzyme modules (A and R) in substrate interaction remain unclear.

Purpose of the Study:

• To investigate the molecular interactions between alginate and AlgE enzymes using dynamic force spectroscopy.
• To elucidate the structure-function relationship of AlgE enzyme modules (A and R) in alginate binding and epimerization.
• To determine the binding strength and kinetics of AlgE-alginate interactions.

Main Methods:

• Single-molecule force spectroscopy (dynamic force spectroscopy) was employed.
• Interactions between alginate and various AlgE constructs (AlgE4, AlgE6, PKA1, A-module, R-module) were measured.
• Protein-mannuronan interaction forces and potential widths were quantified.

Main Results:

• The A-module demonstrated direct binding to alginate, with interaction strengths varying by enzyme (e.g., 73 pN for AlgE4, 144 pN for A-module).
• The R-module showed no substrate attraction, indicating it modulates binding rather than directly interacting.
• AlgE4 exhibited significantly longer polymer residence times, suggesting a processive mechanism.

Conclusions:

• The A-module of AlgE enzymes is responsible for substrate binding.
• The R-module plays a crucial role in modulating enzyme-substrate binding affinity.
• AlgE4 likely operates via a processive mechanism, distinct from non-processive constructs like PKA1.