The pSUFER analysis revealed severe suboptimal in the labeling of A271 as well as flexible changes in the loop where A271 is located in previous simulations of the WT-D-PL complex and the loop region

The pSUFER analysis has revealed a significant suboptimal labeling of A271, indicating a deficiency in the labeling of this residue. Additionally, previous simulations of the WT-D-PL complex have shown that the loop region where A271 is located exhibits a degree of flexibility. This loop region plays a crucial role in regulating the activity of D-lac. Interestingly, the pSUFER results have also shown that F274, which is situated in the same loop as A271, exhibits more than 10 suboptimal markers, despite being distanced from the substrate by approximately 10 Å. This finding suggests that the distal mutation may have some effect on the enzyme activity.

To address this issue, F308G was selected as a template to mutate F274 to H, K, R, or L. The resulting mutants, namely F274H, F274K, and F274R, were found to be synergistic with F308G, indicating an enhancement in activity. Subsequently, the mutant N96S/A271E/F308G was used as a template to introduce Y at position 274, as it exists in the pSUFER space and has a similar side chain to F. The results have shown that all four generated quadruple mutants exhibited an increase in activity, with the mutant N96S/A271E/F274Y/F308G showing a remarkable 56-fold increase in activity.

It is evident that the identification and optimization of key residues, such as A271 and F274, can significantly improve the activity of D-lac. The findings from this study provide valuable insights into the structure-function relationship of D-lac and pave the way for the development of more efficient biocatalysts