Structure and Function of a Dehydrating Condensation Domain in Nonribosomal Peptide Biosynthesis.

Dehydroamino acids are important structural motifs and biosynthetic intermediates for natural products. Many bioactive natural products of nonribosomal origin contain dehydroamino acids; however, the biosynthesis of dehydroamino acids in most nonribosomal peptides is not well understood. Here, we provide biochemical and bioinformatic evidence in support of the role of a unique class of condensation domains in dehydration (CmodAA). We also obtain the crystal structure of a CmodAA domain, which is part of the nonribosomal peptide synthetase AmbE in the biosynthesis of the antibiotic methoxyvinylglycine. Biochemical analysis reveals that AmbE-CmodAA modifies a peptide substrate that is attached to the donor carrier protein. Mutational studies of AmbE-CmodAA identify several key residues for activity, including four residues that are mostly conserved in the CmodAA subfamily. Alanine mutation of these conserved residues either significantly increases or decreases AmbE activity. AmbE exhibits a dimeric conformation, which is uncommon and could enable transfer of an intermediate between different protomers. Our discovery highlights a central dehydrating function for CmodAA domains that unifies dehydroamino acid biosynthesis in diverse nonribosomal peptide pathways. Our work also begins to shed light on the mechanism of CmodAA domains. Understanding CmodAA domain function may facilitate identification of new natural products that contain dehydroamino acids and enable engineering of dehydroamino acids into nonribosomal peptides.

Biosynthesis of fluopsin C, a copper-containing antibiotic from Pseudomonas aeruginosa.

Metal-binding natural products contribute to metal acquisition and bacterial virulence, but their roles in metal stress response are underexplored. We show that a five-enzyme pathway in Pseudomonas aeruginosa synthesizes a small-molecule copper complex, fluopsin C, in response to elevated copper concentrations. Fluopsin C is a broad-spectrum antibiotic that contains a copper ion chelated by two minimal thiohydroxamates. Biosynthesis of the thiohydroxamate begins with cysteine and requires two lyases, two iron-dependent enzymes, and a methyltransferase. The iron-dependent enzymes remove the carboxyl group and the α carbon from cysteine through decarboxylation, N-hydroxylation, and methylene excision. Conservation of the pathway in P. aeruginosa and other bacteria suggests a common role for fluopsin C in the copper stress response, which involves fusing copper into an antibiotic against other microbes.

Author Correction: Optogenetic control of cofilin and αTAT in living cells using Z-lock.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Optogenetic control of cofilin and αTAT in living cells using Z-lock.

Here we introduce Z-lock, an optogenetic approach for reversible, light-controlled steric inhibition of protein active sites. The light oxygen voltage (LOV) domain and Zdk, a small protein that binds LOV selectively in the dark, are appended to the protein of interest where they sterically block the active site. Irradiation causes LOV to change conformation and release Zdk, exposing the active site. Computer-assisted protein design was used to optimize linkers and Zdk-LOV affinity, for both effective binding in the dark, and effective light-induced release of the intramolecular interaction. Z-lock cofilin was shown to have actin severing ability in vitro, and in living cancer cells it produced protrusions and invadopodia. An active fragment of the tubulin acetylase αTAT was similarly modified and shown to acetylate tubulin on irradiation.