Structural and functional impacts of charge modification in Chondroitinase ABC I

Chondroitinase ABC I from Proteus vulgaris is a glycosaminoglycan lyase with potential for promoting axon regeneration after spinal cord injury (SCI). This enzyme has a minor negative charge at physiological pH. To investigate the impact of surface charge modification, we targeted a surface-exposed helix at the central domain, replacing alanine at position 360 with either glutamate (A360E) or threonine (A360T). Structural models generated using the MODELLER program revealed that neither mutation significantly altered the overall protein structure. However, fluorescence spectroscopy indicated a more compact structure in both mutants compared to the wild-type. Circular dichroism spectroscopy further showed increased stabilizing interactions in the helical content of A360E mutant. Thermal stability analysis showed a higher unfolding enthalpy change for A360E compared to A360T and the wild-type, suggesting greater cooperativity in stabilizing interactions and reduced population of partially denatured states at moderate temperatures. The A360E mutant exhibited enhanced catalytic efficiency compared to both A360T and the wild-type enzymes. These findings suggest that replacing a surface-exposed hydrophobic residue with a negatively charged one (A360E) leads to increased protein stability, likely due to favorable interactions with water dipoles and enhanced solubility through reducing the probability of aggregation via increased intermolecular electrostatic repulsion interactions.