Computational design of noncanonical amino acid-based thioether staples at N/C-terminal domains of multi-modular pullulanase for thermostabilization in enzyme catalysis.

TitleComputational design of noncanonical amino acid-based thioether staples at N/C-terminal domains of multi-modular pullulanase for thermostabilization in enzyme catalysis.
Publication TypeJournal Article
Year of Publication2021
AuthorsBi J, Jing X, Wu L, Zhou X, Gu J, Nie Y, Xu Y
JournalComput Struct Biotechnol J
Volume19
Pagination577-585
Date Published2021
ISSN2001-0370
Abstract

Enzyme thermostabilization is considered a critical and often obligatory step in biosynthesis, because thermostability is a significant property of enzymes that can be used to evaluate their feasibility for industrial applications. However, conventional strategies for thermostabilizing enzymes generally introduce non-covalent interactions and/or natural covalent bonds caused by natural amino acid substitutions, and the trade-off between the activity and stability of enzymes remains a challenge. Here, we developed a computationally guided strategy for constructing thioether staples by incorporating noncanonical amino acid (ncAA) into the more flexible N/C-terminal domains of the multi-modular pullulanase from (BtPul) to enhance its thermostability. First, potential thioether staples located in the N/C-terminal domains of BtPul were predicted using RosettaMatch. Next, eight variants involving stable thioether staples were precisely predicted using FoldX and Rosetta ddg_monomer. Six positive variants were obtained, of which T73(O2beY)-171C had a 157% longer half-life at 70 °C and an increase of 7.0 °C in , when compared with the wild-type (WT). T73(O2beY)-171C/T126F/A72R exhibited an even more improved thermostability, with a 211% increase in half-life at 70 °C and a 44% enhancement in enzyme activity compared with the WT, which was attributed to further optimization of the local interaction network. This work introduces and validates an efficient strategy for enhancing the thermostability and activity of multi-modular enzymes.

DOI10.1016/j.csbj.2020.12.043
Alternate JournalComput Struct Biotechnol J
PubMed ID33510863
PubMed Central IDPMC7811066