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

. 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.