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Updated: Jul 20, 2026
In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
Published on: March 25, 2020
Interdomain communication between the thiolation and thioesterase domains of EntF explored by combinatorial
Zhe Zhou1, Jonathan R Lai, Christopher T Walsh
1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
Abstract:
Thiolation (T) domains are protein way stations in natural product assembly lines. In the enterobactin synthetase, the T domain on EntF is recognized in cis by its catalytic partners: the EntF condensation (C), adenylation (A), and thioesterase (TE) domains. To assess surface features of the EntF T domain recognized by C, A, and TE, regions of the EntF T domain were submitted to shotgun alanine scanning and Ent production selection, which revealed residues that could not be substituted by Ala. EntF mutants bearing Ala in such positions were assayed in vitro for Ent production with EntEB, and for A-T, C-T, and T-TE communications. We concluded that G1027A and M1030A are specifically defective in acyl transfer from T to TE. These residues define an interaction surface between these two in cis domains in an NRPS module.
