Leveraging Substrate Promiscuity of a Radical <i>S</i>-Adenosyl-<scp>L</scp>-methionine RiPP Maturase toward Intramolecular Peptide Cross-Linking Applications

Eastman, Karsten A. S.; Kincannon, William M.; Bandarian, Vahe

doi:10.1021/acscentsci.2c00501

Cited by 16 publications

(30 citation statements)

References 42 publications

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“…We had previously shown that PapB is capable of crosslinking peptides where the Cys and Asp residues are adjacent, as well as those separated by up to six residues. 35 This suggests a model for recognition of the substrate where the active site recognizes the thiol and carboxylate moieties of the substrate only and that the remainder of the substrate, apart from the leader sequence, does not serve a role in recognition. This was further underscored by the ability to replace even the Cys and the Asp residues with D-amino acids and generate thioether cross-links in those substrates.…”

Section: ■ Discussionmentioning

confidence: 99%

“…37 In our previous report, we demonstrated that PapB will cyclize Cys and Asp side chains that are adjacent as well as those separated by up to six residues. 35 Epimeric peptide substrates bearing D-configured Cys and Asp residues are also suitable for cyclization by PapB. Leveraging this tolerance, we generated a thioether containing analog of the FDA-approved drug octreotide, with a Cys-Glu cross-link replacing the disulfide in a threonine capped peptide.…”

Section: ■ Introductionmentioning

confidence: 99%

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A Promiscuous rSAM Enzyme Enables Diverse Peptide Cross-linking

Eastman

Mifflin

Oblad

et al. 2023

ACS Bio Med Chem Au

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Ribosomally produced and post-translationally modified polypeptides (RiPPs) are a diverse group of natural products that are processed by a variety of enzymes to their biologically relevant forms. PapB is a member of the radical S-adenosyl-l-methionine (rSAM) superfamily that introduces thioether cross-links between Cys and Asp residues in the PapA RiPP. We report that PapB has high tolerance for variations in the peptide substrate. Our results demonstrate that branched side chains in the thiol- and carboxylate-containing residues are processed and that lengthening of these groups to homocysteine and homoglutamate does not impair the ability of PapB to form thioether cross-links. Remarkably, the enzyme can even cross-link a peptide substrate where the native Asp carboxylate moiety is replaced with a tetrazole. We show that variations to residues embedded between the thiol- and carboxylate-containing residues are tolerated by PapB, as peptides containing both bulky (e.g., Phe) and charged (e.g., Lys) side chains in both natural L- and unnatural D-forms are efficiently cross-linked. Diastereomeric peptides bearing (2S,3R)- and (2S,3S)-methylaspartate are processed by PapB to form cyclic thioethers with markedly different rates, suggesting the enzymatic hydrogen atom abstraction event for the native Asp-containing substrate is diastereospecific. Finally, we synthesized two diastereomeric peptide substrates bearing E- and Z-configured γ,δ-dehydrohomoglutamate and show that PapB promotes addition of the deoxyadenosyl radical (dAdo•) instead of hydrogen atom abstraction. In the Z-configured γ,δ-dehydrohomoglutamate substrate, a fraction of the dAdo-adduct peptide is thioether cross-linked. In both cases, there is evidence for product inhibition of PapB, as the dAdo-adducts likely mimic the native transition state where dAdo• is poised to abstract a substrate hydrogen atom. Collectively, these findings provide critical insights into the arrangement of reacting species in the active site of the PapB, reveal unusual promiscuity, and highlight the potential of PapB as a tool in the development peptide therapeutics.

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Section: ■ Discussionmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

A Promiscuous rSAM Enzyme Enables Diverse Peptide Cross-linking

Eastman

Mifflin

Oblad

et al. 2023

ACS Bio Med Chem Au

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“…The previously reported correction factor for the Bradford assays was used in determining concentration and stoichiometry. 49 Amino Acid Analysis and Iron Concentration Determination of Tte1186. The iron content of WT Tte1186 as well as the Fe−S cluster knockouts was established as documented previously.…”

Section: ■ Experimental Proceduresmentioning

confidence: 99%

Peptide Selenocysteine Substitutions Reveal Direct Substrate–Enzyme Interactions at Auxiliary Clusters in Radical S-Adenosyl-l-methionine Maturases

et al. 2023

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Radical S-adenosyl-l-methionine (SAM) enzymes leverage the properties of one or more iron- and sulfide-containing metallocenters to catalyze complex and radical-mediated transformations. By far the most populous superfamily of radical SAM enzymes are those that, in addition to a 4Fe–4S cluster that binds and activates the SAM cofactor, also bind one or more additional auxiliary clusters (ACs) of largely unknown catalytic significance. In this report we examine the role of ACs in two RS enzymes, PapB and Tte1186, that catalyze formation of thioether cross-links in ribosomally synthesized and post-translationally modified peptides (RiPPs). Both enzymes catalyze a sulfur-to-carbon cross-link in a reaction that entails H atom transfer from an unactivated C–H to initiate catalysis, followed by formation of a C–S bond to yield the thioether. We show that both enzymes tolerate substitution of SeCys instead of Cys at the cross-linking site, allowing the systems to be subjected to Se K-edge X-ray spectroscopy. The EXAFS data show a direct interaction with the Fe of one of the ACs in the Michaelis complex, which is replaced with a Se–C interaction under reducing conditions that lead to the product complex. Site-directed deletion of the clusters in Tte1186 provide evidence for the identity of the AC. The implications of these observations in the context of the mechanism of these thioether cross-linking enzymes are discussed.

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“… 10 , 11 Similarly, RiPP enzymes possess a recognition element that can be used to guide a defined leader sequence to different active sites for transformation. 90 Driven by the high reactivity of the initial Ado· radicals and subsequent substrate promiscuity that this can entail, 91 a huge range of possibilities for new types of transformation is opened up if different active sites can be paired with different recognition elements in a modular fashion.…”

Section: Manipulating the Architecturementioning

confidence: 99%

“…Nature has already demonstrated this modular construction with some of the newly structurally characterized B 12 -AdoMet radical enzymes, which have discrete but integrated motifs to carry out dual chemistries. , Similarly, RiPP enzymes possess a recognition element that can be used to guide a defined leader sequence to different active sites for transformation . Driven by the high reactivity of the initial Ado· radicals and subsequent substrate promiscuity that this can entail, a huge range of possibilities for new types of transformation is opened up if different active sites can be paired with different recognition elements in a modular fashion.…”

Section: Manipulating the Architecturementioning

confidence: 99%

If It Is Hard, It Is Worth Doing: Engineering Radical Enzymes from Anaerobes

Jäger

Croft

2022

Biochemistry

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With a pressing need for sustainable chemistries, radical enzymes from anaerobes offer a shortcut for many chemical transformations and deliver highly sought-after functionalizations such as late-stage C–H functionalization, C–C bond formation, and carbon-skeleton rearrangements, among others. The challenges in handling these oxygen-sensitive enzymes are reflected in their limited industrial exploitation, despite what they may deliver. With an influx of structures and mechanistic understanding, the scope for designed radical enzymes to deliver wanted processes becomes ever closer. Combined with new advances in computational methods and workflows for these complex systems, the outlook for an increased use of radical enzymes in future processes is exciting.

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Leveraging Substrate Promiscuity of a Radical S-Adenosyl-L-methionine RiPP Maturase toward Intramolecular Peptide Cross-Linking Applications

Cited by 16 publications

References 42 publications

A Promiscuous rSAM Enzyme Enables Diverse Peptide Cross-linking

A Promiscuous rSAM Enzyme Enables Diverse Peptide Cross-linking

Peptide Selenocysteine Substitutions Reveal Direct Substrate–Enzyme Interactions at Auxiliary Clusters in Radical S-Adenosyl-l-methionine Maturases

If It Is Hard, It Is Worth Doing: Engineering Radical Enzymes from Anaerobes

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