the dDistal mutation may have some effect on the enzyme activity 58-62 So F308G was then selected as a template to mutate F274 to H K R or L The F274H F274K and F274R mutants were also found to be syn

The dDistal mutation appears to exert some influence on enzyme activity between positions 58-62. In light of this, F308G was selected as a template for introducing mutations at position F274, with H, K, R, or L being considered as potential substitutions. Remarkably, F274H, F274K, and F274R mutants were found to act synergistically with F308G (as depicted in Figure 4F). Encouraged by these findings, the N96S/A271E/F308G mutant was utilized as a framework for introducing further substitutions at position F274, with Y being chosen due to its presence in the pSUFER space and its side chain similarity to F. The results demonstrated that the activity of all four quadruple mutants was enhanced, with the N96S/A271E/F274Y/F308G mutant exhibiting a remarkable 56-fold increase in activity (as illustrated in Figure 4F)