Directed Evolution Reveals the Functional Sequence Space of an Adenylation Domain Specificity Code
Abstract
Nonribosomal peptides are important natural products biosynthesized by nonribosomal peptide synthetases (NRPSs). Adenylation (A) domains of NRPSs are highly specific for the substrate they recognize. This recognition is determined by 10 residues in the substrate-binding pocket, termed the specificity code. This finding led to the proposal that nonribosomal peptides could be altered by specificity code swapping. Unfortunately, this approach has proven, with few exceptions, to be unproductive; changing the specificity code typically results in broadened specificity or poor function. To enhance our understanding of A domain substrate selectivity, we carried out a detailed analysis of the specificity code from the A domain of EntF, an NRPS involved in enterobactin biosynthesis in Escherichia coli. Using directed evolution and a genetic selection, we determined which sites in the code have strict residue requirements and which are tolerant of variation. We showed that the EntF A domain, and other l-Ser-specific A domains, have a functional sequence space for l-Ser recognition, rather than a single code. This functional space is more expansive than the aggregate of all characterized l-Ser-specific A domains: we identified 152 new l-Ser specificity codes. Together, our data provide essential insights into how to overcome the barriers that prevent rational changesmore »
- Authors:
-
[1]
- Univ. of Wisconsin, Madison, WI (United States)
- Pennsylvania State Univ., University Park, PA (United States)
- Publication Date:
- Research Org.:
- Univ. of Wisconsin, Madison, WI (United States). Great Lakes Bioenergy Research Center
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER); National Science Foundation (NSF); National Institutes of Health (NIH)
- OSTI Identifier:
- 1601235
- Grant/Contract Number:
- SC0018409; NSF1716594; GM100346
- Resource Type:
- Accepted Manuscript
- Journal Name:
- ACS Chemical Biology
- Additional Journal Information:
- Journal Volume: 14; Journal Issue: 9; Journal ID: ISSN 1554-8929
- Publisher:
- American Chemical Society (ACS)
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; peptides and proteins; genetics; monomers; chemical specificity; energy; directed evolution; siderophore; substrate specificity; nonribosomal peptide synthetase; adenylation domain; natural product
Citation Formats
Throckmorton, Kurt, Vinnik, Vladimir, Chowdhury, Ratul, Cook, Taylor, Chevrette, Marc G., Maranas, Costas, Pfleger, Brian, and Thomas, Michael George. Directed Evolution Reveals the Functional Sequence Space of an Adenylation Domain Specificity Code. United States: N. p., 2019.
Web. doi:10.1021/acschembio.9b00532.
Throckmorton, Kurt, Vinnik, Vladimir, Chowdhury, Ratul, Cook, Taylor, Chevrette, Marc G., Maranas, Costas, Pfleger, Brian, & Thomas, Michael George. Directed Evolution Reveals the Functional Sequence Space of an Adenylation Domain Specificity Code. United States. https://doi.org/10.1021/acschembio.9b00532
Throckmorton, Kurt, Vinnik, Vladimir, Chowdhury, Ratul, Cook, Taylor, Chevrette, Marc G., Maranas, Costas, Pfleger, Brian, and Thomas, Michael George. Tue .
"Directed Evolution Reveals the Functional Sequence Space of an Adenylation Domain Specificity Code". United States. https://doi.org/10.1021/acschembio.9b00532. https://www.osti.gov/servlets/purl/1601235.
@article{osti_1601235,
title = {Directed Evolution Reveals the Functional Sequence Space of an Adenylation Domain Specificity Code},
author = {Throckmorton, Kurt and Vinnik, Vladimir and Chowdhury, Ratul and Cook, Taylor and Chevrette, Marc G. and Maranas, Costas and Pfleger, Brian and Thomas, Michael George},
abstractNote = {Nonribosomal peptides are important natural products biosynthesized by nonribosomal peptide synthetases (NRPSs). Adenylation (A) domains of NRPSs are highly specific for the substrate they recognize. This recognition is determined by 10 residues in the substrate-binding pocket, termed the specificity code. This finding led to the proposal that nonribosomal peptides could be altered by specificity code swapping. Unfortunately, this approach has proven, with few exceptions, to be unproductive; changing the specificity code typically results in broadened specificity or poor function. To enhance our understanding of A domain substrate selectivity, we carried out a detailed analysis of the specificity code from the A domain of EntF, an NRPS involved in enterobactin biosynthesis in Escherichia coli. Using directed evolution and a genetic selection, we determined which sites in the code have strict residue requirements and which are tolerant of variation. We showed that the EntF A domain, and other l-Ser-specific A domains, have a functional sequence space for l-Ser recognition, rather than a single code. This functional space is more expansive than the aggregate of all characterized l-Ser-specific A domains: we identified 152 new l-Ser specificity codes. Together, our data provide essential insights into how to overcome the barriers that prevent rational changes to A domain specificity.},
doi = {10.1021/acschembio.9b00532},
journal = {ACS Chemical Biology},
number = 9,
volume = 14,
place = {United States},
year = {Tue Aug 20 00:00:00 EDT 2019},
month = {Tue Aug 20 00:00:00 EDT 2019}
}
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