Part:BBa_K5319716

Mutation-vioE

vioE activity prediction model was established

---

We used heterogeneous ensemble learning to construct a causal map of enzyme activity contribution from the activity data (including but not limited to molecular properties and energy landscape data) obtained from the two-site saturated mutation library. Then the baseline is constructed by rearranging the graph relations, and the graph model constructed in the previous step is tested. Based on the model that violates the local markov condition least in the second step, the data is recombined, and multiple rounds of one-step bayesian sampling are carried out to generate the expectation-multi-factor distribution map. Further screening and experimental verification of software models Based on this relationship, we were able to screen out the sample group with the greatest possible increase in enzyme activity from the larger virtual mutant library. In the predicted area (pocket size 600±100 or 1200±100, channel length 6±2), mutants with higher enzyme activity were most likely to be obtained (Figure 2), so we conducted further experimental verification and repeated the measurement results twice as shown in Figure 2 (). The correctness of the software was confirmed by experimental results, in which we obtained mutants in the predicted region (pocket size 600±100 or 1200±100, channel length 6±2) that increased enzyme activity up to 32%.

Further screening and experimental verification of software models

---

We performed MD simulations on Glu-His mutants to elucidate the molecular mechanism of increased enzyme activity. Compared with WT-ligand, the mutant significantly enlarged the substrate binding pocket (Figure 2 a,b). The larger substrate-binding pocket reduces the steric hindrance for ligand binding to the enzyme, thus facilitating the catalytic reaction. The Rg value of the mutant complex was significantly greater than the Rg value of the wild-type (WT) protein, indicating that after the substrate was docked with the mutant protein, the protein chain expanded and the enzyme activity increased relative to the WT protein (Figure 2 c, d). Calculation of the number of hydrogen bonds showed that the mutant complex formed more hydrogen bonds than the WT protein, thereby increasing the rate of the catalytic reaction (Figure 2 e, f). Furthermore, we noticed an altered charge distribution in the mutant substrate binding pocket (Figure 2 g,h). This results in a reduction in the number of positive charges in the active center and a shift in charge effect to neutrality. Improve the enzyme activity of vioE by eliminating the influence of electrostatic interactions.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 578
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 484
  
• 1000
  COMPATIBLE WITH RFC[1000]