Reconstitution of Iterative Thioamidation in Closthioamide Biosynthesis Reveals Tailoring Strategy for Nonribosomal Peptide Backbones

Abstract

Thioamide-containing nonribosomal peptides (NRPs) are exceedingly rare.R ecently the biosynthetic gene cluster for the thioamidated NRP antibiotic closthioamide (CTA) was reported, however,the enzyme responsible for and the timing of thioamide formation remained enigmatic. Here, genome editing, biochemical assays,and mutational studies are used to demonstrate that an Fe-S cluster containing member of the adenine nucleotide a-hydrolase protein superfamily (CtaC) is responsible for sulfur incorporation during CTAbiosynthesis.H owever,u nlike all previously characterized members, CtaC functions in at hiotemplated manner.I na ddition to prompting ar evision of the CTAb iosynthetic pathway, the reconstitution of CtaC provides the first example of aN RP thioamide synthetase.Finally,CtaC is used as abioinformatic handle to demonstrate that thioamidated NRP biosynthetic gene clusters are more widespread than previously appreciated.

Reconstitution of polythioamide antibiotic backbone formation reveals unusual thiotemplated assembly strategy

“…end-capped β-alanine (βAla) polymers attached to the peptidyl carrier protein (PCP) CtaE (5,6). However, as all of the biosynthetic intermediates identified to date from both wild-type and knockout strains contain thioamides (5)(6)(7)14), the origin of this PHBA-βAla conjugate was a mystery.…”

“…end-capped β-alanine (βAla) polymers attached to the peptidyl carrier protein (PCP) CtaE (5,6). However, as all of the biosynthetic intermediates identified to date from both wild-type and knockout strains contain thioamides (5)(6)(7)14), the origin of this PHBA-βAla conjugate was a mystery. Further complicating matters, the biosynthetic enzymes expected to be responsible for forming this CtaE-linked intermediate-CtaD, CtaG, and CtaI-belong to protein families more commonly associated with primary metabolism than with NRP assembly, making the a priori prediction of their substrates difficult.…”

“…The CTA biosynthetic locus also encodes a second PCP (Fig. 1B), CtaH, with no proven role in CTA assembly (5,6). Given the conservation of this second PCP in similar biosynthetic gene clusters, we predicted that CtaH would be critical for CTA maturation.…”

“…However, a notable exception came with the recent discovery of a thiotemplated yet NRPS-independent pathway for the biosynthesis of closthioamide (CTA; Fig. 1 A and B) (5,6).…”

“…Knockout studies and in vitro biochemical assays led to the identification of the enzymes responsible for sulfur insertion and late-stage intermediate dimerization (Fig. 1C) (5,6). The biosynthetic strategy for assembly of the amide backbone of CTA has remained a mystery, however.…”