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Original Research Article

Review Article


Woodard, A.M.; Peccati, F.; Navo, C.D.; Jiménez-Osés, G.; Mitchell, D.A.; "Darobactin substrate engineering and computation show radical stability governs ether versus C–C bond formation" J. Am. Chem. Soc., (2024) doi.org/10.1021/jacs.4c03994

Investigation of the radical SAM enzyme involved in darobactin biosynthesis. Computational and experimental work provides new insight into darobactin and rSAM catalysis.


Lee, A.R.; Carter R.S.; Imani, A.S.; Dommaraju, S.R.; Hudson, G.A.; Mitchell, D.A.; Freeman, M.F.; "Discovery of borosin catalytic strategies and function through bioinformatic profiling" ACS Chem. Biol., (2024) doi.org/10.1021/acschembio.4c00066

RODEO was expanded to analyze borosins, facilitating a large-scale analysis of borosins. This analysis led to the discovery of several new aspects of borosin biosynthesis


Shi, C.; Patel, V.D.; Mitchell, D.A.; Zhao, H.; "Polythiazole-containing hemolytic peptide from Enterococcus caccae" ChemBioChem, (2024) doi.org/10.1002/cbic.202400212

A streptolysin S-like cytolysin was discovered in Enterococcus allowing for a more detailed structural characterization of an elusive virulence factor


Nguyen, D.T.; Zhu, L.; Gray, D.L.; Woods, T.J.; Padhi, C.; Flatt, K.M.; Mitchell, D.A.; van der Donk, W.A.; "Biosynthesis of macrocyclic peptides with C-terminal β-amino-α-keto acid groups by three different metalloenzymes" ACS Cent. Sci., (2024) doi.org/10.1021/acscentsci.4c00088

Bioinformatics was utilized to discover a novel RiPP class biosynthesized by distinct metalloenzyme families including MNIO, B12-rSAM, and cytochrome P450


Ren, H.; Huang, C.; Pan, Y.; Dommaraju, S.R.; Cui, H.; Li, M.; Gadgil, M.G.; Mitchell, D.A.; Zhao, H.; "Non-modular fatty acid synthases yield distinct N-terminal acylation in ribosomomal peptides" Nat. Chem., (2024) doi.org/10.1038/s41557-024-01491-3

Identified a new compound, “lipoavitide", that is a fatty acid/RiPP hybrid. Using structural characterization and in vitro reconstitution, a putative biosynthetic pathway was suggested.


Harris, L.A.; Saad, H.; Shelton, K.E.; Zhu, L.; Guo, X.; Mitchell, D.A.; "Trytophan-centric bioinformatics identifies new lasso peptide modifications" Biochemistry, 63: 865-879 (2024) doi.org/10.1021/acs.biochem.4c00035

Bioinformatic strategy to discover lasso peptides with new modifications to tryptophan was used to identify and characterize two news groups of lasso peptides.


Nguyen, D.T.; Mitchell, D.A.; van der Donk, W.A.; "Genome mining for new enzyme chemistry" ACS Catal., 14: 4536-4553 (2024) doi.org/10.1021/acscatal.3c06322

A review describing the advances in mining genome for new chemical transformations


Fernandez, H.; Kretsch, A.; Kunakom, S.; Kadjo, A.; Mitchell, D.; Eustaquio, A.; "High-yield lasso peptide production in a Burkholderia bacterial host by plasmid copy number engineering." ACS Synth. Biol., 13: 337-350 (2024) doi.org/10.1021/acssynbio.3c00597

Tuning of plasmid copy number in a Burkholderia isolate was leveraged to produce two new lasso peptides, mycetolassins, in high titers.


Saad, H.; Majer, T.; Bhattarai, K.; Lampe, S.; Nguyen, D.T.; Kramer, M.; Straetenger, J.; Oesterbelt, H.B.; Mitchell, D.A.; Gross, H. "Bioinformatic-guided discovery of biaryl-linked lasso peptides." Chem. Sci., 14: 13176-13183 (2023) doi.org/10.1039/D3SC02380J

Dscovery and structural characterization of two new (C-N) biaryl-tailored lasso peptides modified by P450 enzymes.


Chadwick, G.L.; Joiner A.M.N.; Ramesh, S.; Mitchell, D.A.; Nayak, D.D. "McrD binds asymmetrically to methyl-coenzyme M reductase improving active-site accessibility during assembly." Proc. Natl. Acad. Sci. USA, 120: e2302815120 (2023). doi.org/10.1073/pnas.2302815120

CryoEM reveals the role of McrD in the assembly of methyl-coenzyme M reductase, a ubiquitous enzyme in methanogens and key player in the global carbon cycle.


Ren, H.; Dommaraju, S.R.; Huang, C.; Cui, H.; Pan, Y.; Nesic, M.; Zhu, L.; Sarlah, D.; Mitchell, D.A.; Zhao, H. "Genome mining unveils a class of ribosomal peptides with two amino termini." Nat. Commun., 14: 1624 (2023). doi.org/10.1038/s41467-023-37287-1.

RRE-Finder was used to bioinformatically discover the daptides, new RiPP class featuring two amino termini.


Precord, T.W.; Ramesh, S.; Dommaraju, S.R.; Harris, L.A.; Kille, B.L.; Mitchell, D.A. "Catalytic site proximity profiling for functional unification of sequence-diverse radical S-adenosylmethionine enzymes." ACS Bio. Med. Chem. Au, 3: 240-251 (2023). doi.org/10.1021/acsbiomedchemau.2c00085.

Identification of paraphyletic sactisynthases by profiling of catalytic site proximity residues. A new sactisynthase from S. sparsogenes is reported using this method.


Kretsch, A.M.; Gadgil, M.G.; DiCaprio, A.J.; Barrett, S.E.; Kille, B.L.; Si, Y.; Zhu, L.; Mitchell, D.A. "Peptidase activation by a leader peptide-bound RiPP recognition element." Biochemistry, 62: 956-967 (2023). doi.org/10.1021/acs.biochem.2c00700

Bioinformatic and biochemical techniques unraveled the function of RRE domains in lasso peptide biosynthesis


Shelton, K.E.; Mitchell, D.A. "Bioinformatic prediction and experimental validation of RiPP recognition elements." Meth. Enzymol., 679: 191-233 (2023). doi.org/10.1016/bs.mie.2022.08.050

A review covering bioinformatic methods to predict RiPP recognition elements (RREs) and experimental methods to confirm the role of RREs as leader peptide binders


Rice, A.J.; Pelton, J.M.; Kramer, N.J.; Catlin, D.S.; Nair, S.K.; Pogorelov, T.V.; Mitchell, D.A.; Bowers, A.A. "Enzymatic pyridine aromatization during thiopeptide biosynthesis." J. Am. Chem. Soc., 144: 21116-21124 (2022). doi.org/10.1021/jacs.2c07377

An in-depth mechanistic investigation of the class-defining [4+2] enzyme in thiopeptide and pyritide biosynthesis


Ayikpoe, R.S.; Shi, C.; Battiste, A.J.; Eslami, S.M.; Ramesh, S.; Simon, M.A.; Bothwell, I.R.; Lee, H.; Rice, A.J.; Ren, H.; Tian, Q.; Harris, L.A.; Sarksian, R.; Zhu, L.; Frerk, A.M.; Precord, T.W.; van der Donk, W.A.; Mitchell, D.A.; Zhao, H. "A scalable platform to discover antimicrobials of ribosomal origin." Nat. Commun., 13: 6135 (2022). doi.org/10.1038/s41467-022-33890-w

A robotic system was developed to rapidly refactor RiPP BGCs, which were then expressed in E. coli. Using this method, three antibacterial RiPPs were discovered.


Ongpipattanakul, C.; Desormeaux, E.K.; DiCaprio, A.J.; van der Donk, W.A.; Mitchell, D.A.; Nair, S.K. "Mechanism of action of ribosomally synthesized and post-translationally modified peptides." Chem. Rev., 122: 14722–14814 (2022). doi.org/10.1021/acs.chemrev.2c00210

Comprehensive review of the modes of action of bacterial RiPPs


Nguyen, D.T.; Le, T.T.; Rice, A.J.; Hudson, G.A.; van der Donk, W.A.; Mitchell, D.A. "Accessing diverse pyridine-based macrocyclic peptides by a two-site recognition pathway." J. Am. Chem. Soc., 144: 11263-11269 (2022). doi.org/10.1021/jacs.2c02824

Characterization of a versatile biosynthetic pathway to generate 14- to 68-membered pyridine-based macrocyclic peptides with diverse structures


Harris, L.A.; Mitchell, D.A. "Reactivity-based screening for natural product discovery." Meth. Enzymol., 665: 177-208 (2022). doi.org/10.1016/bs.mie.2021.11.018

A review detailing the use of reactivity-based screening to discover natural products with specific functional groups


Oberg, N.; Precord, T.W.; Mitchell, D.A.; Gerlt, J.A. "RadicalSAM.org: a resource to interpret sequence-function space and discover new radical SAM enzyme chemistry." ACS Bio. Med. Chem. Au, 2: 22-35 (2022). doi.org/10.1021/acsbiomedchemau.1c00048

RadicalSAM.org, a new web-based genomic enzymology resource, is described, which aims to accelerate the characterization of the rSAM superfamily


Ramesh, S.; Guo, X.; DiCaprio, A.J.; De Lio, A.M.; Harris, L.A.; Kille, B.L.; Pogorelov, T.V.; Mitchell, D.A. "Bioinformatics-guided expansion and discovery of graspetides." ACS Chem. Biol., 16: 2787-2797 (2021). doi.org/10.1021/acschembio.1c00672

RODEO's utility was expanded to cover graspetides, facilitating the most comprehensive bioinformatic analysis of graspetides to date and the discovery of conformational isomers thatisin and iso-thatisin


Liu, A.; Krushnamurthy, P.H.; Subramanya, K.S.; Mitchell, D.A.; Mahanta, N. "Enzymatic thioamidation of peptide backbones." Meth. Enzymol., 656: 459-494 (2021). doi.org/10.1016/bs.mie.2021.04.010

A detailed review of methods and precise experimental protocols for investigating peptide backbone thioamidation by YcaO enzymes.


Guo, X.R.; Zhang, J.; Li, X.; Xiao, E.; Lange, J.; Rienstra, C.; Burke, M.D.; Mitchell, D.A. "Sterol sponge mechanism is conserved for glycosylated polyene macrolides." ACS Cent. Sci., 7: 781-791 (2021). doi.org/10.1021/acscentsci.1c00148

Bioinformatics and a tetrazine-based probe were used to expand the glycosylated polyene macrolide natural product class, allowing the confirmation of a generalized sterol sponge mechanism of action.


Si, Y.; Kretsch, A.M.; Daigh, L.M.; Burk, M.J.; Mitchell, D.A. "Cell-Free biosynthesis to evaluate lasso peptide formation and enzyme–substrate tolerance." J. Am. Chem. Soc., 143: 5917–5927 (2021). doi.org/10.1021/jacs.1c01452

Cell-free biosynthesis was used to produce known and novel lasso peptides, and to evaluate enzyme-substrate tolerance.


Liu, A.; Si, Y.; Dong, S.; Mahanta, N.; Penkala, H.N.; Nair, S.K.; Mitchell, D.A. "Functional elucidation of TfuA in peptide backbone thioamidation." Nat. Chem. Biol., 17: 585-592 (2021). doi.org/10.1038/s41589-021-00771-0

Functional revelation of the TfuA protein family and the proteinaceous sulfur donor involved in peptide backbone thioamidation.


Harris, L.A.; Saint-Vincent, P.M.B.; Guo, X.R.; Hudson, G.A.; DiCaprio, A.J.; Zhu, L.; Mitchell, D.A. "Reactivity-based screening for citrulline-containing natural products reveals a family of bacterial peptidyl arginine deiminases." ACS Chem. Biol., 15: 3167–3175 (2020). doi.org/10.1021/acschembio.0c00685

A combination of a citrulline specific probe and other methods was used to discover the family of bacterial PADs responsible for converting arginine to citrulline in citrulassin biosynthesis.


Georgiou, M.A.; Dommaraju, S.R.; Guo, X.R.; Mast, D.H.; Mitchell, D.A. "Bioinformatic and reactivity-based discovery of linaridins." ACS Chem. Biol., 15: 2976-2985 (2020). doi.org/10.1021/acschembio.0c00620

RODEO’s utility was expanded to cover all linaridins, this showed a wide diversity in the subclass and lead to the discovery of pegvadin A and B.


Montalbán-López, M. et al. "New developments in RiPP discovery, enzymology and engineering." Nat. Prod. Rep., 38: 130-239 (2021). doi.org/10.1039/D0NP00027B

An update to Arnison et al. 2013, this review focuses on the advances in the RiPP field from 2013-2020.


Kloosterman, A.M.; Shelton, K.E.; van Wezel, G.P.; Medema, M.H.; Mitchell, D.A. "RRE-Finder: A genome-mining tool for class-independent RiPP discovery." mSystems, 5: e00267-20 (2020). doi.org/10.1128/mSystems.00267-20

The RRE-Finder tool rapidly identifies RiPP recognition elements in gene clusters, facilitating discovery of novel natural products.


Hudson, G.A.; Hooper, A.R.; DiCaprio, A.J.; Sarlah, D.; Mitchell, D.A. "Structure prediction and synthesis of pyridine-based macrocyclic peptide natural products." Org. Lett., 23: 253–256 (2021). doi.org/10.1021/acs.orglett.0c02699

The product of a biosynthetic gene cluster was structurally predicted, synthesized, and verified by enzyme reconstitution, requiring a reclassification of thiopeptides as a subclass of the pyritides.


Walker, M.C.; Eslami, S.M.; Hetrick, K.J.; Ackenhusen, S.E.; Mitchell, D.A.; van der Donk, W.A. "Precursor peptide-targeted mining of more than one hundred thousand genomes expands the lanthipeptide natural product family." BMC Genomics, 21: 387-403 (2020). doi.org/10.1186/s12864-020-06785-7

RODEO’s utility was expanded to covered all lanthipeptides, revealing the hidden size an diversity of this molecular class while also facilitating the discovery of birimositide.


Nayak, D.D.; Liu, A.; Agrawal, N.; Rodriguez-Carerro, R.; Dong, S.H.; Mitchell, D.A.; Nair, S.K.; Metcalf, W.W. "Functional interactions between posttranslationally modified amino acids of methyl-coenzyme M reductase in Methanosarcina acetivorans." PLoS Biol., 18: e3000507 (2020). doi.org/10.1371/journal.pbio.3000507

Characterization of three posttranslational modifications on methyl-coenzyme M reductase reveals their complex interaction, serving to fine-tune the enzyme activity.


Sieber, S.; Grendelmeier, S.M.; Harris, L.A.; Mitchell, D.A.; Gademann, K. "Microviridin 1777: A toxic chymotrypsin inhibitor discovered by a metabolomic approach." J. Nat. Prod., 83: 438-446 (2020). doi.org/10.1021/acs.jnatprod.9b00986

A novel chymotrypsin inhibitor, microviridin 1777, was structurally characterized and found to be cytotoxic towards the grazer Thamnocephalus platyurus.