An Engineered Aryl Acid Adenylation Domain with an Enlarged Substrate Binding Pocket
- 3 April 2019
- journal article
- research article
- Published by Wiley in Angewandte Chemie-International Edition
- Vol. 58 (21), 6906-6910
- https://doi.org/10.1002/anie.201900318
Abstract
Adenylation (A) domains act as the gatekeepers of non‐ribosomal peptide synthetases (NRPSs), ensuring the activation and thioesterification of the correct amino acid/aryl acid building blocks. Aryl acid building blocks are most commonly observed in iron‐chelating siderophores, but are not limited to them. Very little is known about the reprogramming of aryl acid A‐domains. We show that a single asparagine‐to‐glycine mutation in an aryl acid A‐domain leads to an enzyme that tolerates a wide range of non‐native aryl acids. The engineered catalyst is capable of activating non‐native aryl acids functionalized with nitro, cyano, bromo, and iodo groups, even though no enzymatic activity of wild‐type enzyme was observed toward these substrates. Co‐crystal structures with non‐hydrolysable aryl‐AMP analogues revealed the origins of this expansion of substrate promiscuity, highlighting an enlargement of the substrate binding pocket of the enzyme. Our findings may be exploited to produce diversified aryl acid containing natural products and serve as a template for further directed evolution in combinatorial biosynthesis.Keywords
Funding Information
- Japan Society for the Promotion of Science (17H05438, 17K07747, 16H06451)
- Takeda Science Foundation
This publication has 29 references indexed in Scilit:
- Precursor‐Directed Diversification of Cyclic Tetrapeptidic PseudoxylallemycinsChemBioChem, 2018
- Nonribosomal biosynthesis of backbone-modified peptidesNature Chemistry, 2017
- Recent advances in engineering nonribosomal peptide assembly linesNatural Product Reports, 2015
- Biosynthesis of Novel Pyoverdines by Domain Substitution in a Nonribosomal Peptide Synthetase of Pseudomonas aeruginosaApplied and Environmental Microbiology, 2014
- Reprogramming Nonribosomal Peptide Synthetases for “Clickable” Amino AcidsAngewandte Chemie, 2014
- Reprogramming Nonribosomal Peptide Synthetases for “Clickable” Amino AcidsAngewandte Chemie-International Edition, 2014
- Explorations of catalytic domains in non-ribosomal peptide synthetase enzymologyNatural Product Reports, 2012
- Introduction of a Non‐Natural Amino Acid into a Nonribosomal Peptide Antibiotic by Modification of Adenylation Domain SpecificityAngewandte Chemie-International Edition, 2012
- Introduction of a Non‐Natural Amino Acid into a Nonribosomal Peptide Antibiotic by Modification of Adenylation Domain SpecificityAngewandte Chemie, 2012
- Nonribosomal Peptide Synthetases Involved in the Production of Medically Relevant Natural ProductsMolecular Pharmaceutics, 2008