Abstract
Objectives
Taxoid 10β-O-acetyl transferase (DBAT) was redesigned to enhance its catalytic activity and substrate preference for baccatin III and taxol biosynthesis.
Results
Residues H162, D166 and R363 were determined as potential sites within the catalytic pocket of DBAT for molecular docking and site-directed mutagenesis to modify the activity of DBAT. Enzymatic activity assays revealed that the kcat/KM values of mutant H162A/R363H, D166H, R363H, D166H/R363H acting on 10-deacetylbaccatin III were about 3, 15, 26 and 60 times higher than that of the wild type of DBAT, respectively. Substrate preference assays indicated that these mutants (H162A/R363H, D166H, R363H, D166H/R363H) could transfer acetyl group from unnatural acetyl donor (e.g. vinyl acetate, sec-butyl acetate, isobutyl acetate, amyl acetate and isoamyl acetate) to 10-deacetylbaccatin III.
Conclusion
Taxoid 10β-O-acetyl transferase mutants with redesigned active sites displayed increased catalytic activities and modified substrate preferences, indicating their possible application in the enzymatic synthesis of baccatin III and taxol.
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Acknowledgements
This work was supported by the Science and Technology Program of Guangdong Province (Grant 2014B050505018, 2014B020205003, 2015A020209121) and the National Natural Science Foundation of China (Grant 31071837, 31572178, 31772373).
Supporting information
Supplementary Table 1—Primers used in the experiments.
Supplementary Table 2—DBAT homology model.
Supplementary Fig. 1—Expression of mutants.
Supplementary Fig. 2—The kinetic curves for DBAT.
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You, LF., Huang, JJ., Wei, T. et al. Enhanced catalytic activities and modified substrate preferences for taxoid 10β-O-acetyl transferase mutants by engineering catalytic histidine residues. Biotechnol Lett 40, 1245–1251 (2018). https://doi.org/10.1007/s10529-018-2573-9
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DOI: https://doi.org/10.1007/s10529-018-2573-9