A copper-containing antibiotic
Bacteria require transition metal ions for biological processes and must also protect themselves against excess metal, which is toxic. Patteson
et al
. explored how the environmental bacterium
Pseudomonas aeruginosa
uses a five-enzyme pathway to synthesize a small-molecule complex, fluopsin C, which is built from cysteine and contains a copper ion. The biosynthesis involves unusual enzymatic transformations that convert cysteine to a thiohydroximate, two of which chelate a copper ion in the final natural product. Fluopsin C protects
P. aeruginosa
from excess copper and also acts as a broad-spectrum antibiotic against other bacteria. —VV
Here we introduce Z-lock, an optogenetic approach for reversible, light-controlled steric inhibition of protein active sites. The 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 upon irradiation.
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.
In the version of this article originally published, several images were incorrectly set. In Supplementary Fig. 1d, the gels for cofilin and actin were switched. The associated full gels, correct in the original submission, are in Supplementary Fig. 3. In Fig. 4b, the bands for "αTAT core" and for "DN αTAT" were stretched when copied from the original gel to the figure. The original and corrected versions of Fig. 4b are shown below. The errors have been corrected in the HTML and PDF versions of the paper and in the Supplementary Information file.
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