Biology subjects

Link, A. J.

Publications and source records attributed to Link, A. J..

3 recordsLinked to original sources

bioRxiv2022

The Anti-Burkholderia Lasso Peptide Ubonodin Co-Opts the Siderophore Receptor PupB for Cellular Entry

New antibiotics are needed as bacterial infections continue to be a leading cause of death. Notorious among antibiotic-resistant bacteria is the Burkholderia cepacia complex (Bcc), which infects cystic fibrosis patients, causing lung function decline. We recently discovered a novel ribosomally synthesized and post-translationally modified peptide (RiPP), ubonodin, with potent activity against several Burkholderia pathogens. Ubonodin inhibits RNA polymerase, but only select Bcc strains were susceptible, indicating that having a conserved cellular target does not guarantee activity. Given the cytoplasmic target, we speculate that cellular uptake of ubonodin determines susceptibility. Here, we report a new outer membrane siderophore receptor, PupB, that is required for ubonodin uptake in B. cepacia. Loss of PupB renders B. cepacia resistant to ubonodin, whereas expressing PupB sensitizes a resistant strain. Thus, outer membrane transport is the major determinant of ubonodins spectrum of activity. We also show that PupB is activated by a TonB protein and examine a transcriptional pathway that further regulates PupB. Finally, we elucidate the complete cellular uptake pathway for ubonodin by also identifying its inner membrane transporter in B. cepacia. Our work unravels central steps in the mechanism of action of ubonodin and establishes a general framework for dissecting RiPP function.

microbiology↗

bioRxiv2021

Cellulonodin-2 and Lihuanodin: Lasso Peptides with an Aspartimide Post-Translational Modification

Lasso peptides are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) defined by their threaded structure. Besides the class-defining isopeptide bond, other post-translational modifications (PTMs) that further tailor lasso peptides have been previously reported. Using genome mining tools, we identified a subset of lasso peptide biosynthetic gene clusters (BGCs) that are colocalized with protein L-isoaspartyl methyltransferase (PIMT) homologs. PIMTs have an important role in protein repair, restoring isoaspartate residues formed from asparagine deamidation to aspartate. Here we report a new function for PIMT enzymes in the post-translational modification of lasso peptides. The PIMTs associated with lasso peptide BGCs first methylate an L-aspartate sidechain found within the ring of the lasso peptide. The methyl ester is then converted into a stable aspartimide moiety, endowing the lasso peptide ring with rigidity relative to its unmodified counterpart. We describe the heterologous expression and structural characterization of two examples of aspartimide-modified lasso peptides from thermophilic Gram-positive bacteria. The lasso peptide cellulonodin-2 is encoded in the genome of actinobacterium Thermobifida cellulosilytica, while lihuanodin is encoded in the genome of firmicute Lihuaxuella thermophila. Additional genome mining revealed PIMT-containing lasso peptide BGCs in 48 organisms. In addition to heterologous expression, we have reconstituted PIMT-mediated aspartimide formation in vitro, showing that lasso peptide-associated PIMTs transfer methyl groups very rapidly as compared to canonical PIMTs. Furthermore, in stark contrast to other characterized lasso peptide PTMs, the methyltransferase functions only on lassoed substrates. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/444711v1_ufig1.gif" ALT="Figure 1"> View larger version (9K): org.highwire.dtl.DTLVardef@158f0d2org.highwire.dtl.DTLVardef@2a6baeorg.highwire.dtl.DTLVardef@103a91dorg.highwire.dtl.DTLVardef@153dd99_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Fuscimiditide: a RiPP with {Omega}-Ester and Aspartimide Post-translational Modifications

Microviridins and other {omega}-ester linked peptides (OEPs) are characterized by sidechain-sidechain linkages installed by ATP-grasp enzymes. Here we describe the discovery of a new family of OEPs, the gene clusters of which also encode an O-methyltransferase with homology to the protein repair catalyst protein L-isoaspartyl methyltransferase (PIMT). We produced the first example of this new ribosomally synthesized and post-translationally modified peptide (RiPP), fuscimiditide, via heterologous expression. NMR analysis of fuscimiditide revealed that the peptide contains two ester crosslinks forming a stem-loop macrocycle. Furthermore, an unusually stable aspartimide moiety is found within the loop macrocycle. We have also fully reconstituted fuscimiditide biosynthesis in vitro establishing that ester formation catalyzed by the ATP-grasp enzyme is an obligate, rate-limiting first biosynthetic step. Aspartimide formation from aspartate is catalyzed by the PIMT homolog in the second step. The aspartimide moiety embedded in fuscimiditide hydrolyzes regioselectively to isoaspartate (isoAsp). Surprisingly, this isoAsp-containing protein is also a substrate for the PIMT homolog, thus driving any hydrolysis products back to the aspartimide form. Whereas aspartimide is often considered a nuisance product in protein formulations, our data here suggest that some RiPPs have aspartimide residues intentionally installed via enzymatic activity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/444834v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1c67f36org.highwire.dtl.DTLVardef@c9e774org.highwire.dtl.DTLVardef@17a7d62org.highwire.dtl.DTLVardef@bde61b_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗