Active site groove volume may influence the DD-carboxypeptidase activity of E. coli DacD

DacD is an enzyme that belongs to the DD-carboxypeptidase family that remove the terminal D-alanine from the peptidoglycan precursor in bacterial cell walls. However, even though it performs this same kind of reaction, its enzymatic efficiency is lower than that of other DD-carboxypeptidases. In order to investigate this, we developed and examined a computer-generated 3D model of DacD, evaluating the capacity of the active-site groove to accommodate certain substrate binding. The volume of the active-site groove in DacD was discovered to be approximately one-third of the predicted value for optimum activity, which may be correlated with reduced catalytic effectiveness. To further investigate this hypothesis, we deliberately induced both transitional and transversional mutations in DacD in silico. These mutations were specifically targeted at secondary amino acid residues located in the vicinity of the active site. The alterations resulted in a 70% increase in the volume of the groove in DacD. Aligning the alpha carbons at the catalytic site of the altered DacD showed a similar geometric layout to that linked with increased activity, indicating that the mutant residues contribute to improved orientation of the active site. Molecular docking, MD simulation, MMGBSA and residue decomposition analysis demonstrated that the mutant DacD has a stronger substrate-binding capacity. These results suggest that the larger volume of the active-site groove leads to a more effective substrate binding. Therefore, we anticipate that a larger groove volume might lead to a greater DD-carboxypeptidase activity, which nevertheless requires further wet laboratory validation.