Biology subjects

Koos, J. D.

Publications and source records attributed to Koos, J. D..

3 recordsLinked to original sources

bioRxiv2023

A large-scale bioinformatic study of graspimiditides and structural characterization of albusimiditide

Graspetides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) that exhibits an impressive diversity in patterns of side chain-to-side chain {omega}-ester or {omega}-amide linkages. Recent studies have uncovered a significant portion of graspetides to contain an additional post-translational modification involving aspartimidylation catalyzed by an O-methyltransferase, predominantly found in the genomes of Actinomycetota. Here, we present a comprehensive bioinformatic analysis focused on graspetides harboring aspartimide for which we propose the name graspimiditides. From Protein BLAST results of 5,000 methyltransferase sequences, we identified 962 unique putative graspimiditides, which we further classified into eight main clusters based on sequence similarity along with several smaller clusters and singletons. The previously studied graspimiditides, fuscimiditide and amycolimiditide, are identified in this analysis; fuscimiditide is a singleton while amycolimiditide is in the fifth largest cluster. Cluster 1, by far the largest cluster, contains 641 members, encoded almost exclusively in the Streptomyces genus. To characterize an example of a graspimiditide in Cluster 1, we conducted experimental studies on the peptide from Streptomyces albus J1074, which we named albusimiditide. By tandem mass spectrometry, hydrazinolysis, and amino acid substitution experiments, we elucidated the structure of albusimiditide to be a large tetracyclic peptide with four {omega}-ester linkages generating a stem-loop structure with one aspartimide. The ester crosslinks form 22-, 46-, 22-, and 44-atom macrocycles, last of which, the loop, contains the enzymatically installed aspartimide. Further in vitro experiments revealed that the aspartimide hydrolyzes in a 3:1 ratio of isoaspartate to aspartate residues. Overall, this study offers a comprehensive insight into the diversity and structural features of graspimiditides, paving the way for future investigations of this unique class of natural product. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/545981v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@bd68f2org.highwire.dtl.DTLVardef@1350705org.highwire.dtl.DTLVardef@5ff593org.highwire.dtl.DTLVardef@1f3c4a9_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

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↗