Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

Simultaneous Improvement of the Thermostability and Catalytic Activity of Epoxidase Lsd18 for the Synthesis of Lasalocid A

Version 1 : Received: 2 November 2023 / Approved: 2 November 2023 / Online: 3 November 2023 (09:15:02 CET)

A peer-reviewed article of this Preprint also exists.

Liu, N.; Xiao, H.; Zang, Y.; Zhou, L.; Mencius, J.; Yang, Z.; Quan, S.; Chen, X. Simultaneous Improvement in the Thermostability and Catalytic Activity of Epoxidase Lsd18 for the Synthesis of Lasalocid A. Int. J. Mol. Sci. 2023, 24, 16795. Liu, N.; Xiao, H.; Zang, Y.; Zhou, L.; Mencius, J.; Yang, Z.; Quan, S.; Chen, X. Simultaneous Improvement in the Thermostability and Catalytic Activity of Epoxidase Lsd18 for the Synthesis of Lasalocid A. Int. J. Mol. Sci. 2023, 24, 16795.

Abstract

Enzymes used in the synthesis of natural products are potent catalysts, capable of efficient and stereoselective chemical transformations. Lsd18 catalyzes two sequential epoxidations during the biosynthesis of lasalocid A, a polyether polyketide natural product. We performed protein engineering on Lsd18 to improve its thermostability and catalytic activity. Utilizing structure-guided methods of FoldX and Rosetta-ddG, we designed 15 mutants of Lsd18. Screening of these mutants using thermal shift assay identified stabilized variants Lsd18-T189M, Lsd18-S195M, and the double mutant Lsd18-T189M-S195M. Trypsin digestion, molecular dynamic simulation, circular dichroism (CD) spectroscopy, and X-ray crystallography provided insights into the molecular basis for the improved enzyme properties. Notably, enhanced hydrophobic interaction within the enzyme core and interaction of the protein with the FAD cofactor appear to be responsible for better thermostability.

Keywords

natural product; Lsd18; thermostability; FoldX; Rossetta-ddG; catalytic activity

Subject

Biology and Life Sciences, Biophysics

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