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.

Siderophores are iron-chelating molecules produced by microorganisms and plants to acquire exogenous iron. Siderophore biosynthetic enzymology often produces elaborate and unique molecules through unusual reactions to enable specific recognition by the producing organisms. Herein, we report the structure of two siderophore analogs from Agrobacterium fabrum strain C58, which we named fabrubactin (FBN) A and FBN B. Additionally, we characterized the substrate specificities of the NRPS and PKS components. The structures suggest unique Favorskii-like rearrangements of the molecular backbone that we propose are catalyzed by the flavin-dependent monooxygenase, FbnE. FBN A and B contain a 1,1-dimethyl-3-amino-1,2,3,4-tetrahydro-7,8-dihydroxy-quinolin (Dmaq) moiety previously seen only in the anachelin cyanobacterial siderophores. We provide evidence that Dmaq is derived from l-DOPA and propose a mechanism for the formation of the mature Dmaq moiety. Our bioinformatic analyses suggest that FBN A and B and the anachelins belong to a large and diverse siderophore family widespread throughout the Rhizobium/Agrobacterium group, α-proteobacteria, and cyanobacteria.

Non‐ribosomal peptides (NRPs) are a medically and agriculturally important class of natural products found in bacteria and fungi that are biosynthesized by non‐ribosomal peptide synthetases (NRPSs). Adenylation (A) domains of NRPSs have a high degree of specificity for the substrate they recognize and are therefore referred to as the gatekeepers of NRPS enzymology. The specificity of A domains is determined by ten residues in the substrate‐binding pocket; thus, these residues are known as the A domain specificity code. To produce new NRPs, efforts have been made to rationally alter A domain specificity by changing the specificity code, but these experiments have typically resulted in proteins with broadened specificity or poor function. We hypothesize that an NRPS‐based genetic selection system is needed to better understand the specificity code by allowing us to identify functional A domains with altered specificity codes in a high‐throughput manner. We used directed evolution to examine the flexibility of the specificity code of EntF from the enterobactin (ENT) siderophore biosynthetic system in E. coli. Proper assembly of ENT requires L‐Ser incorporation and thereby provides a genetic selection for functional L‐Ser recognition. We created several entF A domain libraries, each randomizing different but overlapping subsets of five specificity code residues using NNK‐containing primers. These libraries were used to complement an E. coli ΔentF mutant in trans, selecting for functional A domain variants on iron‐limiting media. We determined that certain positions in the specificity code have strict requirements, while others are tolerant of substitutions and often contain residues not observed in known L‐Ser codes. All the isolated EntF variants retain specificity solely for L‐Ser and have biological and kinetic activity comparable to that of wildtype EntF. This work expands our understanding of A domain specificity and will aid in engineering of NRPSs to produce new natural products.Support or Funding InformationThis work is supported by the National Science Foundation grant 1716594.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.