Mechanistic Analysis of the Biosynthesis of the Aspartimidylated Graspetide Amycolimiditide

Choi, Brian; Elashal, Hader E.; Cao, Li; Link, A. James

doi:10.1021/jacs.2c09004

Cited by 10 publications

(39 citation statements)

References 40 publications

(108 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As introduced above, the hydrolysis of lihuanodin is regioselective to give Asp and no isoAsp (Figure c). This is in stark contrast to the hydrolysis of aspartimides formed by canonical PIMTs as well as in fuscimiditide, amycolimiditide, and OlvA(BCS A ), which are regioselective favoring isoAsp. − , We further confirmed this result by treating hydrolyzed lihuanodin with human PIMT, which will convert isoAsp (and not Asp) to aspartimide, , and observed no reaction (Figure a). As we showed previously, the hydrolyzed material is quantitatively converted to lihuanodin upon LihM treatment.…”

mentioning

confidence: 57%

“…Protein isoaspartyl methyltransferases (PIMTs) make up a family of enzymes that reverse damage in aging proteins. − PIMTs restore function of proteins that have accumulated isoaspartyl residues by driving their conversion to the l -aspartyl form . PIMTs are highly conserved and are found in almost all eukaryotic cells and Gram-negative bacteria. , In Gram-positive bacterial genomes, recent work by our group as well as van der Donk and co-workers has illustrated that enzymes homologous to PIMTs are associated with several ribosomally synthesized and post-translationally modified peptide (RiPP) biosynthetic gene clusters (BGCs), , specifically in the BGCs of lasso peptides cellulonodin-2 and lihuanodin (Figure a,b), graspetides fuscimiditide and amycolimiditide, , and lanthipeptide OlvA(BCS A ) . The PIMT homologues in cellulonodin-2 and lihuanodin BGCs, TceM and LihM, respectively, recognize matured lasso peptides pre-cellulonodin-2 and pre-lihuanodin as substrates , and methylate the first aspartate residue in a conserved DTAD motif in the lasso peptide ring, using S -adenosylmethionine (SAM) (Figure c) .…”

mentioning

confidence: 99%

See 1 more Smart Citation

Kinetics of Aspartimide Formation and Hydrolysis in Lasso Peptide Lihuanodin

2023

Self Cite

View full text Add to dashboard Cite

Aspartimides are notorious as undesired side products in solid-phase peptide synthesis and in pharmaceutical formulations. However, we have discovered several ribosomally synthesized and post-translationally modified peptides (RiPPs) in which aspartimide is installed intentionally via enzymatic activity of protein L-isoaspartyl methyltransferase (PIMT) homologues. In the case of the lasso peptide lihuanodin, the methyltransferase LihM recognizes the lassoed substrate pre-lihuanodin, specifically methylating the side chain of an L-Asp residue in the ring portion of the lasso peptide. The subsequent nucleophilic attack from the adjacent amide leads to the formation of an aspartimide. The resulting aspartimide hydrolyzes regioselectively to L-Asp in buffers above pH 7. Here we report the first Michaelis−Menten kinetic measurements of such a RiPP-associated PIMT homologue, LihM, acting on its cognate substrate pre-lihuanodin. Additionally, we measured the rate of aspartimide hydrolysis, which allowed us to deduce the kinetics of the entire reaction network. The relative magnitudes of these rates explain the accumulation and relative stability of aspartimide-containing lihuanodin. We also demonstrate that the residue C-terminal to the aspartimide controls the regioselectivity of hydrolysis and thus the threadedness of the peptide.

show abstract

mentioning

confidence: 57%

mentioning

confidence: 99%

Kinetics of Aspartimide Formation and Hydrolysis in Lasso Peptide Lihuanodin

2023

Self Cite

View full text Add to dashboard Cite

show abstract

“…We named this putative imiditide mNmaA M following the nomenclature used in our study on amycolimiditide, where m stands for modified, and superscripted M stands for the methyltransferase. 21 Our observation indicated that the rate of methylation was faster than the rate of aspartimidylation for the biosynthesis of mNmaA M (Figure S6). Since NmaM is homologous to PIMT, we wonder whether Pcm, the housekeeping PIMT in E. coli, could alter the intended product in vivo.…”

Section: Heterologous Expression Of Imiditide From Nonomuraea Maritimamentioning

confidence: 71%

“…Both amycolimiditide and pre-amycolimiditide have 4 ester linkages, but amycolimiditide has an additional aspartimide moiety suggested by the solution NMR structure (PDB: 8DYM). 21 Indeed, both spectra showed a peak at 1742 cm -1 , indicating the presence of esters. Additionally, the FTIR spectrum for amycolimiditide also contains an additional peak at 1708 cm -1 , indicating this peak corresponds to an aspartimide formation (Figure S12).…”

Section: Mnmaa M Contains An Aspartimide Moietymentioning

confidence: 94%

“…Our laboratory has previously characterized several RiPPs with aspartimide posttranslational modifications, including lasso peptides cellulonodin-2 and lihuanodin, 15 as well as graspetides fuscimiditide and amycolimiditide. 17,21 In their biosynthesis, a dedicated Omethyltransferase related to protein isoaspartyl methyltransferases (PIMT) installs the aspartimide on the matured lasso peptides or graspetides as the last step of the reaction series. Since PIMT homologs cluster with various families of RiPPs (Figure 1a), we reason that these PIMT homologs can serve as a bioinformatic handle to unveil uncharacterized families of RiPPs.…”

Section: Identification Of Biosynthetic Gene Clusters For a Putative ...mentioning

confidence: 99%

See 1 more Smart Citation

Genome Mining and Discovery of Imiditides, a Novel Family of RiPPs with a Class-defining Aspartimide Modification

Cao

Zhu

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a fascinating class of natural products of ribosomal origins. In the past decade, various sophisticated machine learning-based software packages have been established to discover novel RiPPs that do not resemble the known families. Instead, we argue that tailoring enzymes that cluster with various RiPP families can serve as effective bioinformatic seeds for novel RiPP discovery. Leveraging that O-methyltransferases homologous to protein isoaspartyl methyltransferases (PIMTs) are associated with lasso peptide, graspetide, and lanthipeptide biosynthetic gene clusters (BGCs), we utilized the C-terminal motif unique to RiPP-associated O-methyltransferases as the search query to discover a novel family of RiPPs, imiditides. Our genome-mining algorithm reveals a total of 670 imiditide BGCs, widely distributed in Gram-positive bacterial genomes. In addition, we demonstrate the heterologous production of the founding member of the imiditide family, mNmaAM, encoded in the genome of Nonomuraea maritima. In contrast to other RiPP associated PIMTs that recognize constrained peptides as substrates, the PIMT homolog in mNmaAM BGC, NmaM, methylates a specific Asp residue on the linear precursor peptide, NmaA. The methyl ester is then turned into an aspartimide spontaneously. The aspartimide moiety formed is unusually stable, leading to the accumulation of the aspartimidylated product in vivo. The substrate specificity is achieved by extensive charge-charge interactions between the precursor NmaA and the modifying enzyme NmaM suggested by both experimental validations as well as an AlphaFold model prediction. Our study suggests that PIMT-mediated aspartimide formation is an underappreciated backbone modification strategy in RiPP biosynthesis, compared to the well-studied backbone rigidification chemistries, such as thiazol(in)e and oxazol(in)e formations. Additionally, our findings suggest that aspartimide formation in Gram-positive bacterial proteomes are not limited to spontaneous protein aging and degradation.

show abstract

Evolutionary Spread of Distinct O‐methyltransferases Guides the Discovery of Unique Isoaspartate‐Containing Peptides, Pamtides

Lee,

Park,

Kim

et al. 2023

Advanced Science

View full text Add to dashboard Cite

Ribosomally synthesized and post‐translationally modified peptides (RiPPs) are a structurally diverse class of natural products with a distinct biosynthetic logic, the enzymatic modification of genetically encoded precursor peptides. Although their structural and biosynthetic diversity remains largely underexplored, the identification of novel subclasses with unique structural motifs and biosynthetic pathways is challenging. Here, it is reported that peptide/protein L‐aspartyl O‐methyltransferases (PAMTs) present in several RiPP subclasses are highly homologous. Importantly, it is discovered that the apparent evolutionary transmission of the PAMT gene to unrelated RiPP subclasses can serve as a basis to identify a novel RiPP subclass. Biochemical and structural analyses suggest that homologous PAMTs convert aspartate to isoaspartate via aspartyl‐O‐methyl ester and aspartimide intermediates, and often require cyclic or hairpin‐like structures for modification. By conducting homology‐based bioinformatic analysis of PAMTs, over 2,800 biosynthetic gene clusters (BGCs) are identified for known RiPP subclasses in which PAMTs install a secondary modification, and over 1,500 BGCs where PAMTs function as a primary modification enzyme, thereby defining a new RiPP subclass, named pamtides. The results suggest that the genome mining of proteins with secondary biosynthetic roles can be an effective strategy for discovering novel biosynthetic pathways of RiPPs through the principle of “guilt by association”.

show abstract

Mechanistic Analysis of the Biosynthesis of the Aspartimidylated Graspetide Amycolimiditide

Cited by 10 publications

References 40 publications

Kinetics of Aspartimide Formation and Hydrolysis in Lasso Peptide Lihuanodin

Kinetics of Aspartimide Formation and Hydrolysis in Lasso Peptide Lihuanodin

Genome Mining and Discovery of Imiditides, a Novel Family of RiPPs with a Class-defining Aspartimide Modification

Evolutionary Spread of Distinct O‐methyltransferases Guides the Discovery of Unique Isoaspartate‐Containing Peptides, Pamtides

Contact Info

Product

Resources

About