P450-Modified Ribosomally Synthesized Peptides with Aromatic Cross-Links

Hu, Yi Ling; Yin, Fang Zhou; Shi, Jing; Ma, Shi Ying; Wang, Zi Ru; Tan, Ren Xiang; Jiao, Rui Hua; Ge, Hui Ming

doi:10.1021/jacs.3c07416

Cited by 14 publications

(4 citation statements)

References 44 publications

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“…Notably, the data set appears to fall off precipitously in size at approximately 1000 Å 3 . This cutoff likely results from the low frequency of larger RiPPs and other bacteriocins in the data set; we now know from bioinformatic efforts that there are many more RiPP gene clusters in bacterial genomes than RiPP natural products that have been isolated and reported in the literature. , Furthermore, a general trend of increasing cLogP is observed as the volume increases.…”

Section: Resultsmentioning

confidence: 99%

Cheminformatics-Guided Cell-Free Exploration of Peptide Natural Products

Pelton,

Hochuli,

Sadecki

et al. 2024

J. Am. Chem. Soc.

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There have been significant advances in the flexibility and power of in vitro cell-free translation systems. The increasing ability to incorporate noncanonical amino acids and complement translation with recombinant enzymes has enabled cell-free production of peptide-based natural products (NPs) and NP-like molecules. We anticipate that many more such compounds and analogs might be accessed in this way. To assess the peptide NP space that is directly accessible to current cell-free technologies, we developed a peptide parsing algorithm that breaks down peptide NPs into building blocks based on ribosomal translation logic. Using the resultant data set, we broadly analyze the biophysical properties of these privileged compounds and perform a retrobiosynthetic analysis to predict which peptide NPs could be directly synthesized in augmented cell-free translation reactions. We then tested these predictions by preparing a library of highly modified peptide NPs. Two macrocyclases, PatG and PCY1, were used to effect the head-to-tail macrocyclization of candidate NPs. This retrobiosynthetic analysis identified a collection of high-priority building blocks that are enriched throughout peptide NPs, yet they had not previously been tested in cell-free translation. To expand the cell-free toolbox into this space, we established, optimized, and characterized the flexizyme-enabled ribosomal incorporation of piperazic acids. Overall, these results demonstrate the feasibility of cell-free translation for peptide NP total synthesis while expanding the limits of the technology. This work provides a novel computational tool for exploration of peptide NP chemical space, that could be expanded in the future to allow design of ribosomal biosynthetic pathways for NPs and NP-like molecules.

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Section: Resultsmentioning

confidence: 99%

Cheminformatics-Guided Cell-Free Exploration of Peptide Natural Products

Pelton,

Hochuli,

Sadecki

et al. 2024

J. Am. Chem. Soc.

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“…To the best of our knowledge, ApyD is the first enzyme reported to methylate the Cβ of Tyr. In contrast, ApyO catalyzes biaryl cross-linking between two Tyr residues, a reaction that has been observed with the non-RiPP natural products arylomycin (AryC) and mycocyclosin (CYP121), albeit with different substrates and stereochemical outcomes. , C–C bond formations involving two aromatic rings have been reported to be catalyzed by cytochrome P450 enzymes in RiPP biosynthesis, ,,, with a recent example (SlyP) catalyzing a similar cross-link as ApyO but without information on atropisomerism . Some of the precursor peptides in the identified BGCs here have other aromatic residues in the second and fourth positions from the C-terminus (Trp/Tyr; see supplementary data set 1), suggesting that other biaryl linkages will be formed.…”

Section: Discussionmentioning

confidence: 99%

Biosynthesis of Macrocyclic Peptides with C-Terminal β-Amino-α-keto Acid Groups by Three Different Metalloenzymes

Nguyen,

Zhu,

Gray

et al. 2024

ACS Cent. Sci.

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Advances in genome sequencing and bioinformatics methods have identified a myriad of biosynthetic gene clusters (BGCs) encoding uncharacterized molecules. By mining genomes for BGCs containing a prevalent peptide-binding domain used for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), we uncovered a new compound class involving modifications installed by a cytochrome P450, a multinuclear iron-dependent non-heme oxidative enzyme (MNIO, formerly DUF692), a cobalamin-and radical Sadenosyl-L-methionine-dependent enzyme (B12-rSAM), and a methyltransferase. All enzymes were functionally expressed in Burkholderia sp. FERM BP-3421. Structural characterization demonstrated that the P450 enzyme catalyzed the formation of a biaryl C−C cross-link between two Tyr residues with the B12-rSAM generating β-methyltyrosine. The MNIO transformed a C-terminal Asp residue into aminopyruvic acid, while the methyltransferase acted on the β-carbon of this α-keto acid. Exciton-coupled circular dichroism spectroscopy and microcrystal electron diffraction (MicroED) were used to elucidate the stereochemical configuration of the atropisomer formed upon biaryl cross-linking. To the best of our knowledge, the MNIO featured in this pathway is the first to modify a residue other than Cys. This study underscores the utility of genome mining to isolate new macrocyclic RiPPs biosynthesized via previously undiscovered enzyme chemistry.

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“…Another workflow involving precursor peptide prediction using RBS analysis was utilized to discover new P450-catalyzed transformations. , Common RBS motifs were first gathered from 10 bacterial species, followed by compiling genome neighborhoods for all bacterial P450 proteins. Any BGCs containing short peptides encoded downstream of a detected RBS were prioritized.…”

Section: Using Prevalent Elements In Ripp Biosynthesis As Bioinformat...mentioning

confidence: 99%

Genome Mining for New Enzyme Chemistry

Nguyen,

Mitchell,

van der Donk

2024

ACS Catal.

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A revolution in the field of biocatalysis has enabled scalable access to compounds of high societal values using enzymes. The construction of biocatalytic routes relies on the reservoir of available enzymatic transformations. A review of uncharacterized proteins predicted from genomic sequencing projects shows that a treasure trove of enzyme chemistry awaits to be uncovered. This Review highlights enzymatic transformations discovered through various genome mining methods and showcases their potential future applications in biocatalysis.

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P450-Modified Ribosomally Synthesized Peptides with Aromatic Cross-Links

Cited by 14 publications

References 44 publications

Cheminformatics-Guided Cell-Free Exploration of Peptide Natural Products

Cheminformatics-Guided Cell-Free Exploration of Peptide Natural Products

Biosynthesis of Macrocyclic Peptides with C-Terminal β-Amino-α-keto Acid Groups by Three Different Metalloenzymes

Genome Mining for New Enzyme Chemistry

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