Aromatic-Mediated Carbohydrate Recognition in Processive Serratia marcescens Chitinases

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Processive glycoside hydrolases are key enzymes for breaking down complex polysaccharides.
• These enzymes feature aromatic residues within their catalytic tunnels, suggesting specific functional roles.
• The precise function of these aromatic residues in substrate binding and processivity remains largely uncharacterized.

Purpose of the Study:

• To investigate the roles of specific aromatic residues in Serratia marcescens chitinases ChiA and ChiB.
• To determine how mutations in these residues affect enzyme dynamics, substrate binding, and overall functionality.
• To elucidate the contribution of local environment and position to the function of aromatic residues.

Main Methods:

• Utilized molecular simulation and free energy calculations to analyze active site dynamics and ligand binding.
• Employed isothermal titration calorimetry to quantify enthalpic and entropic contributions to binding free energy.
• Generated site-directed variants of ChiA and ChiB, focusing on six key aromatic residues (Trp and Phe).

Main Results:

• Point mutations of tryptophan residues to alanine resulted in decreased binding free energy compared to wild-type enzymes.
• Residues located at the entrances of substrate-binding clefts showed the most significant negative impact upon mutation, correlating with reduced processivity.
• Phenylalanine mutations exhibited minimal effects on chito-oligomer binding and chitinase functionality.

Conclusions:

• Specific tryptophan residues, particularly those at cleft entrances, are essential for efficient substrate binding and processivity in chitinases.
• Phenylalanine residues appear to play a less critical role in substrate binding compared to tryptophan residues in these enzymes.
• The findings highlight the context-dependent importance of aromatic residues in the catalytic machinery of processive glycoside hydrolases.