Structure of an engineered intein reveals thiazoline ring and provides mechanistic insight
| Autor: | Marlene Belfort, Jing Zhang, Binbin Liu, Georges Belfort, Reza Nemati, Zhong Li, Dan Fabris, Matteo Scalabrin, C. Seth Pearson, Hongmin Li |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
0106 biological sciences
0301 basic medicine Molecular switch biology Stereochemistry Thiazoline Active site Bioengineering Ring (chemistry) 01 natural sciences Applied Microbiology and Biotechnology 03 medical and health sciences chemistry.chemical_compound 030104 developmental biology chemistry Tetrahedral carbonyl addition compound Protein splicing 010608 biotechnology RNA splicing biology.protein Intein Biotechnology |
| Zdroj: | Biotechnology and Bioengineering. 116:709-721 |
| ISSN: | 0006-3592 |
| DOI: | 10.1002/bit.26875 |
| Popis: | We have engineered an intein which spontaneously and reversibly forms a thiazoline ring at the native N-terminal Lys-Cys splice junction. We identified conditions to stablize the thiazoline ring and provided the first crystallographic evidence, at 1.54 A resolution, for its existence at an intein active site. The finding bolsters evidence for a tetrahedral oxythiazolidine splicing intermediate. In addition, the pivotal mutation maps to a highly conserved B-block threonine, which is now seen to play a causative role not only in ground-state destabilization of the scissile N-terminal peptide bond, but also in steering the tetrahedral intermediate toward thioester formation, giving new insight into the splicing mechanism. We demonstrated the stability of the thiazoline ring at neutral pH as well as sensitivity to hydrolytic ring opening under acidic conditions. A pH cycling strategy to control N-terminal cleavage is proposed, which may be of interest for biotechnological applications requiring a splicing activity switch, such as for protein recovery in bioprocessing. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Plný text