Improving enzyme activity of glucosamine-6-phosphate synthase by semi-rational design strategy and computer analysis
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ORIGINAL RESEARCH PAPER
Improving enzyme activity of glucosamine-6-phosphate synthase by semi-rational design strategy and computer analysis Piwu Li . Kang Li . Xu Li . Fei Zhao . Ruiming Wang . Junqing Wang
Received: 10 November 2019 / Accepted: 24 June 2020 Ó Springer Nature B.V. 2020
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 computeraided 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 Piwu Li and Kang Li, the two first authors, contributed equally to this work.
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10529-020-02949-3) contains supplementary material, which is available to authorized users. P. Li R. Wang J. Wang (&) State Key Laboratory of Biobased Material and Green Papermaking (LBMP) (Qilu University of Technology), Jinan 250353, Shandong, People’s Republic of China e-mail: [email protected] P. Li K. Li X. Li F. Zhao R. Wang J. Wang Key Laboratory of Shandong Microbial Engineering, QILU University of Technology (Shandong Academy of Sciences), Jinan 250353, Shandong, People’s Republic of China
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. Keywords Glucosamine-6-phosphate synthase Enzyme activity Computer simulation Site saturation mutagenesis Molecular dynamics simulation Abbreviations BsGlms Glucosamine-6-phosphate synthase from B. subtilis 168 GATase Glutamine aminotransferase BsGlmsThe L593 amino acid of BsGlms L593S protein mutated into the S593 amino acid BsGlmsThe A594 amino acid of BsGlms A594G protein mutated into the G594 amino acid BsGlmsThe K595 amino acid of BsGlms K595H protein mutated into the H595 amino acid BsGlmsThe S596 amino acid of BsGlms S596F protein mutated into the F596 amino acid
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Biotechnol Lett
BsGlmsV597R F6P G6P BsGlms-F6P BsGlmsL593S-F6P BsGlmsA594G-F6P BsGlmsK595H-F6P BsGlmsS596F-F6P BsGlmsV597R-F6P
The V597 amino acid of BsGlms protein mutated into the R597 amino acid Fructose 6-phosphate Glucose 6-phosphate Docking complex of BsGlms and F6P Docking complex of BsGlms-L593S and F6P Docking complex of BsGlms-A594G and F6P Docking complex of BsGlms-K595H and F6P Docking complex of BsGlms-S596F and F6P Docking complex of BsGlms-V597R and F6P
Introduction Glucos
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