Abstract
Objective
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.
Results
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.
Conclusion
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.
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Abbreviations
- BsGlms:
-
Glucosamine-6-phosphate synthase from B. subtilis 168
- GATase:
-
Glutamine aminotransferase
- BsGlms-L593S:
-
The L593 amino acid of BsGlms protein mutated into the S593 amino acid
- BsGlms-A594G:
-
The A594 amino acid of BsGlms protein mutated into the G594 amino acid
- BsGlms-K595H:
-
The K595 amino acid of BsGlms protein mutated into the H595 amino acid
- BsGlms-S596F:
-
The S596 amino acid of BsGlms protein mutated into the F596 amino acid
- BsGlms-V597R:
-
The V597 amino acid of BsGlms protein mutated into the R597 amino acid
- F6P:
-
Fructose 6-phosphate
- G6P:
-
Glucose 6-phosphate
- BsGlms-F6P:
-
Docking complex of BsGlms and F6P
- BsGlms-L593S-F6P:
-
Docking complex of BsGlms-L593S and F6P
- BsGlms-A594G-F6P:
-
Docking complex of BsGlms-A594G and F6P
- BsGlms-K595H-F6P:
-
Docking complex of BsGlms-K595H and F6P
- BsGlms-S596F-F6P:
-
Docking complex of BsGlms-S596F and F6P
- BsGlms-V597R-F6P:
-
Docking complex of BsGlms-V597R and F6P
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Acknowledgements
This work was supported by the National Science Foundation of China (31801527), Taishan industry leading talent (tscy20180103), Shandong Provincial Natural Science Foundation (ZR2016CB04), Major Program of National Natural Science Foundation of Shandong (ZR2017ZB0208), Major Science and Technology Projects in Shandong Province (2016CYJS07A01), Cultivation Project of Shandong Synthetic Biotechnology Innovation Center (sdsynbio-2018-PY-02) and Focus on Research and Development Plan in Shandong Province (2018YFJH0401).
Supporting information
Supplementary Table 1—Primers used in this study.
Supplementary Fig. 1—aPCR product of bsglms gene. M: marker DL5000; 1-4:1803 bp DNA fragment of bsglms gene from Bacillus subtilis 168. b The plasmid electrophoresis pattern of the pET-28a (+) plasmid double-digested with Ncol and Xhol. M: marker DL5000; 1-4:5251 bp double-digested fragment.
Supplementary Fig. 2—The simulation curve was drawn after MD simulation of rmsd of BsGlms-L593S (red) and BsGlms (blue) within 10 ns. At 6 ns to 10 ns, the value of RMSD remains essentially unchanged, indicating that the protein is in a stable state.
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Li, P., Li, K., Li, X. et al. Improving enzyme activity of glucosamine-6-phosphate synthase by semi-rational design strategy and computer analysis. Biotechnol Lett 42, 2319–2332 (2020). https://doi.org/10.1007/s10529-020-02949-3
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DOI: https://doi.org/10.1007/s10529-020-02949-3