Directed Evolution Reveals the Functional Sequence Space of an Adenylation Domain Specificity Code

Kurt Throckmorton, Vladimir Vinnik, Ratul Chowdhury, Taylor Cook, Marc G. Chevrette, Costas Maranas, Brian Pfleger, Michael George Thomas

Research output: Contribution to journalArticlepeer-review

15 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)2044-2054
Number of pages11
JournalACS chemical biology
Issue number9
StatePublished - Sep 20 2019

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Medicine


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