Improving enzyme activity of glucosamine-6-phosphate synthase by semi-rational design strategy and computer analysis

To improve enzyme activity of Glucosamine-6-phosphate synthase (Glms) of Bacillus subtilis by site saturation mutagenesis at Leu₅₉₃, Ala₅₉₄, Lys₅₉₅, Ser₅₉₆ and Val₅₉₇ based on computer-aided semi-rational design.

The results indicated that L₅₉₃S had the greatest effect on the activity of BsGlms and the enzyme activity increased from 5 to 48 U/mL. The mutation of L₅₉₃S increased the yield of glucosamine by 1.6 times that of the original strain. The binding energy of the mutant with substrate was reduced from - 743.864 to - 768.246 kcal/mol. Molecular dynamics simulation results showed that Ser₅₉₃ enhanced the flexibility of the protein, which ultimately led to increased enzyme activity.

We successfully improved BsGlms activity through computer simulation and site saturation mutagenesis. This combination of methodologies may fit into an efficient workflow for improving Glms and other proteins activity.