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Engineered reactivity of a bacterial E1-like enzyme enables ATP-driven modification of protein and peptide C termini

Nature Chemistry volume 17, pages 1371–1382 (2025)Cite this article

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Abstract

In biological systems, adenosine triphosphate (ATP) provides an energetic driving force for peptide bond formation, but protein chemists lack tools that emulate this strategy. Here we develop an ATP-driven platform for C-terminal activation and peptide ligation based on MccB, a bacterial ancestor of ubiquitin-activating (E1) enzymes. We show that MccB can act on non-native substrates to generate an O-AMPylated electrophile that reacts with exogenous nucleophiles to form diverse C-terminal functional groups including thioesters, a versatile class of biological intermediates that have been exploited for protein C-terminal bioconjugation. By mining the natural diversity of the MccB family, we identify both epitope-specific and more promiscuous MccBs. We show that epitope-specific MccB activity can be directed toward specific proteins of interest to enable high-yield, ATP-driven protein bioconjugation, and promiscuous MccB activity can be deployed for the synthesis of peptide thioester substrates for bioconjugation. Our method mimics the chemical logic of biological peptide bond synthesis for high-yield in vitro manipulation of protein structure with molecular precision.

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Fig. 1: MccB catalyses C-terminal O-AMPylation of non-native substrates.
Fig. 2: MccB catalyses the formation of a peptidyl-O-AMP intermediate that can react with exogenous nucleophiles.
Fig. 3: Fusion of the TeCH-tag to proteins enables MccB-catalysed, ATP-dependent formation of C-terminal thioesters.
Fig. 4: Natural MccA/MccB diversity encompasses orthogonal enzymes for C-terminal protein modification.
Fig. 5: MccB enables ATP-dependent thioester formation and regeneration for high-yield enzyme-catalysed EPL.
Fig. 6: Enzymatic synthesis of Ub-derived peptide thioesters using an MccB homologue with relaxed substrate specificity.

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Data availability

All data supporting the conclusions of this paper can be found in the main text, Supplementary Information or Dryad repository at https://doi.org/10.5061/dryad.c59zw3rkb (ref. 66). Source data are provided with this paper.

References

  1. Gomez, M. A. R. & Ibba, M. Aminoacyl-tRNA synthetases. RNA 26, 910–936 (2020).

    Article  CAS  Google Scholar 

  2. Cane, D. E., Walsh, C. T. & Khosla, C. Harnessing the biosynthetic code: combinations, permutations and mutations. Science 282, 63–68 (1998).

    Article  CAS  PubMed  Google Scholar 

  3. Schulman, B. A. & Harper, J. W. Ubiquitin-like protein activation by E1 enzymes: the apex for downstream signalling pathways. Nat. Rev. Mol. Cell Biol. 10, 319–331 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Walsh, C. T., Tu, B. P. & Tang, Y. Eight kinetically stable but thermodynamically activated molecules that power cell metabolism. Chem. Rev. 118, 1460–1494 (2018).

    Article  CAS  PubMed  Google Scholar 

  5. Westheimer, F. H. Why nature chose phosphates. Science 235, 1173–1178 (1987).

    Article  CAS  PubMed  Google Scholar 

  6. Thompson, R. E. & Muir, T. W. Chemoenzymatic semisynthesis of proteins. Chem. Rev. 120, 3051–3126 (2020).

    Article  CAS  PubMed  Google Scholar 

  7. Nguyen, G. K. T. et al. Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis. Nat. Chem. Biol. 10, 732–738 (2014).

    Article  CAS  PubMed  Google Scholar 

  8. Popp, M. W., Antos, J. M., Grotenbreg, G. M., Spooner, E. & Ploegh, H. L. Sortagging: a versatile method for protein labeling. Nat. Chem. Biol. 3, 707–708 (2007).

    Article  CAS  PubMed  Google Scholar 

  9. Popp, M. W. & Ploegh, H. L. Making and breaking peptide bonds: protein engineering using sortase. Angew. Chem. Int. Ed. 50, 5024–5032 (2011).

    Article  CAS  Google Scholar 

  10. Fottner, M. et al. A modular toolbox to generate complex polymeric ubiquitin architectures using orthogonal sortase enzymes. Nat. Commun. 12, 6515 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Fottner, M. et al. Site-specific protein labeling and generation of defined ubiquitin-protein conjugates using an asparaginyl endopeptidase. J. Am. Chem. Soc. 144, 13118–13126 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Fottner, M. et al. Site-specific ubiquitylation and SUMOylation using genetic-code expansion and sortase. Nat. Chem. Biol. 15, 276–284 (2019).

    Article  CAS  PubMed  Google Scholar 

  13. Rehm, F. B. H. et al. Site-specific sequential protein labeling catalyzed by a single recombinant ligase. J. Am. Chem. Soc. 141, 17388–17393 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Abrahmsen, L. et al. Engineering subtilisin and its substrates for efficient ligation of peptide bonds in aqueous solution. Biochemistry 30, 4151–4159 (1991).

    Article  CAS  PubMed  Google Scholar 

  15. Nguyen, G. K. T., Cao, Y., Wang, W., Liu, C. F. & Tam, J. P. Site‐specific N‐terminal labeling of peptides and proteins using butelase 1 and thiodepsipeptide. Angew. Chem. 127, 15920–15924 (2015).

    Article  Google Scholar 

  16. Williamson, D. J., Fascione, M. A., Webb, M. E. & Turnbull, W. B. Efficient N‐terminal labeling of proteins by use of sortase. Angew. Chem. 124, 9511–9514 (2012).

    Article  Google Scholar 

  17. Pihl, R., Zheng, Q. & David, Y. Nature-inspired protein ligation and its applications. Nat. Rev. Chem. 7, 234–255 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Burroughs, A. M., Iyer, L. M. & Aravind, L. Natural history of the E1‐like superfamily: implication for adenylation, sulfur transfer, and ubiquitin conjugation. Proteins Struct. Funct. Bioinform. 75, 895–910 (2009).

    Article  CAS  Google Scholar 

  19. Hochstrasser, M. All in the ubiquitin family. Science 289, 563–564 (2000).

    Article  CAS  PubMed  Google Scholar 

  20. Begley, T. P., Xi, J., Kinsland, C., Taylor, S. & McLafferty, F. The enzymology of sulfur activation during thiamin and biotin biosynthesis. Curr. Opin. Chem. Biol. 3, 623–629 (1999).

    Article  CAS  PubMed  Google Scholar 

  21. Rajagopalan, K. V. Biosynthesis and processing of the molybdenum cofactors. Biochem. Soc. Trans. 25, 757–761 (1997).

    Article  CAS  PubMed  Google Scholar 

  22. Roush, R. F., Nolan, E. M., Löhr, F. & Walsh, C. T. Maturation of an Escherichia coli ribosomal peptide antibiotic by ATP-consuming N-P bond formation in microcin C7. J. Am. Chem. Soc. 130, 3603–3609 (2008).

    Article  CAS  PubMed  Google Scholar 

  23. Regni, C. A. et al. How the MccB bacterial ancestor of ubiquitin E1 initiates biosynthesis of the microcin C7 antibiotic. EMBO J. 28, 1953–1964 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Dikic, I. & Schulman, B. A. An expanded lexicon for the ubiquitin code. Nat. Rev. Mol. Cell Biol. 24, 273–287 (2023).

    Article  CAS  PubMed  Google Scholar 

  25. Komander, D. & Rape, M. The ubiquitin code. Annu. Rev. Biochem. 81, 203–229 (2012).

    Article  CAS  PubMed  Google Scholar 

  26. Guijarro, J. I. et al. Chemical structure and translation inhibition studies of the antibiotic microcin C7. J. Biol. Chem. 270, 23520–23532 (1995).

    Article  CAS  PubMed  Google Scholar 

  27. Metlitskaya, A. et al. Maturation of the translation inhibitor microcin C. J. Bacteriol. 191, 2380–2387 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Kulikovsky, A. et al. The molecular mechanism of aminopropylation of peptide-nucleotide antibiotic microcin C. J. Am. Chem. Soc. 136, 11168–11175 (2014).

    Article  CAS  PubMed  Google Scholar 

  29. Novikova, M. et al. The Escherichia coli Yej transporter is required for the uptake of translation inhibitor microcin C. J. Bacteriol. 189, 8361–8365 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Metlitskaya, A. et al. Aspartyl-tRNA synthetase is the target of peptide nucleotide antibiotic microcin C. J. Biol. Chem. 281, 18033–18042 (2006).

    Article  CAS  PubMed  Google Scholar 

  31. Zukher, I. et al. Reiterative synthesis by the ribosome and recognition of the N-terminal formyl group by biosynthetic machinery contribute to evolutionary conservation of the length of antibiotic microcin C peptide precursor. mBio 10, e00768-19 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Bantysh, O. et al. Enzymatic synthesis of bioinformatically predicted microcin C-like compounds encoded by diverse bacteria. mBio 5, e01059-14 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Webb, M. R. A continuous spectrophotometric assay for inorganic phosphate and for measuring phosphate release kinetics in biological systems. Proc. Natl Acad. Sci. USA 89, 4884–4887 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Lloyd, A. J., Thomann, H.-U., Ibba, M. & Soöll, D. A broadly applicable continuous spectrophotometric assay for measuring aminoacyl-tRNA synthetase activity. Nucleic Acids Res. 23, 2886–2892 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Kazakov, T., Metlitskaya, A. & Severinov, K. Amino acid residues required for maturation, cell uptake and processing of translation inhibitor microcin C. J. Bacteriol. 189, 2114–2118 (2007).

    Article  CAS  PubMed  Google Scholar 

  36. Saha, A. & Deshaies, R. J. Multimodal activation of the ubiquitin ligase SCF by Nedd8 conjugation. Mol. Cell 32, 21–31 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Weeks, A. M. & Chang, M. C. Y. Catalytic control of enzymatic fluorine specificity. Proc. Natl Acad. Sci. USA 109, 19667–19672 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Matthews, M. L. et al. Chemoproteomic profiling and discovery of protein electrophiles in human cells. Nat. Chem. 9, 234–243 (2017).

    Article  CAS  PubMed  Google Scholar 

  39. Pickart, C. M. & Eddins, M. J. Ubiquitin: structures, functions, mechanisms. Biochim. Biophys. Acta Mol. Cell Res. 1695, 55–72 (2004).

    Article  CAS  Google Scholar 

  40. Muir, T. W., Sondhi, D. & Cole, P. A. Expressed protein ligation: a general method for protein engineering. Proc. Natl Acad. Sci. USA 95, 6705–6710 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Fellouse, F. A. et al. High-throughput generation of synthetic antibodies from highly functional minimalist phage-displayed libraries. J. Mol. Biol. 373, 924–940 (2007).

    Article  CAS  PubMed  Google Scholar 

  42. Suzuki, T. et al. Development of cysteine-free fluorescent proteins for the oxidative environment. PLoS ONE 7, e37551 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Dorr, B. M., Ham, H. O., An, C., Chaikof, E. L. & Liu, D. R. Reprogramming the specificity of sortase enzymes. Proc. Natl Acad. Sci. USA 111, 13343–13348 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Podracky, C. J. et al. Laboratory evolution of a sortase enzyme that modifies amyloid-β protein. Nat. Chem. Biol. 17, 317–325 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Henager, S. H. et al. Enzyme-catalyzed expressed protein ligation. Nat. Methods 13, 925–927 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Weeks, A. M. & Wells, J. A. Subtiligase-catalyzed peptide ligation. Chem. Rev. 120, 3127–3160 (2019).

    Article  PubMed  Google Scholar 

  47. Henager, S. H., Henriquez, S., Dempsey, D. R. & Cole, P. A. Analysis of site-specific phosphorylation of PTEN by using enzyme-catalyzed expressed protein ligation. ChemBioChem 21, 64–68 (2020).

    Article  CAS  PubMed  Google Scholar 

  48. Chen, I., Dorr, B. M. & Liu, D. R. A general strategy for the evolution of bond-forming enzymes using yeast display. Proc. Natl Acad. Sci. USA 108, 11399–11404 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Hofmann, R., Akimoto, G., Wucherpfennig, T. G., Zeymer, C. & Bode, J. W. Lysine acylation using conjugating enzymes for site-specific modification and ubiquitination of recombinant proteins. Nat. Chem. 12, 1008–1015 (2020).

    Article  CAS  PubMed  Google Scholar 

  50. Muhar, M. F. et al. C-terminal amides mark proteins for degradation via SCF-FBXO31. Nature 638, 519–527 (2025).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Wang, M. & Casey, P. J. Protein prenylation: unique fats make their mark on biology. Nat. Rev. Mol. Cell Biol. 17, 110–122 (2016).

    Article  CAS  PubMed  Google Scholar 

  52. Clancy, K. W., Melvin, J. A. & McCafferty, D. G. Sortase transpeptidases: insights into mechanism, substrate specificity and inhibition. Pept. Sci. 94, 385–396 (2010).

    Article  CAS  Google Scholar 

  53. Otto, H.-H. & Schirmeister, T. Cysteine proteases and their inhibitors. Chem. Rev. 97, 133–172 (1997).

    Article  CAS  PubMed  Google Scholar 

  54. Kane, P. M. et al. Protein splicing converts the yeast TFP1 gene product to the 69-kDa subunit of the vacuolar H+-adenosine triphosphatase. Science 250, 651–657 (1990).

    Article  CAS  PubMed  Google Scholar 

  55. Novikova, O. et al. Intein clustering suggests functional importance in different domains of life. Mol. Biol. Evol. 33, 783–799 (2016).

    Article  CAS  PubMed  Google Scholar 

  56. Berndsen, C. E. & Wolberger, C. New insights into ubiquitin E3 ligase mechanism. Nat. Struct. Mol. Biol. 21, 301–307 (2014).

    Article  CAS  PubMed  Google Scholar 

  57. Antos, J. M. et al. Site-specific N- and C-terminal labeling of a single polypeptide using sortases of different specificity. J. Am. Chem. Soc. 131, 10800–10801 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Hananya, N., Koren, S. & Muir, T. W. Interrogating epigenetic mechanisms with chemically customized chromatin. Nat. Rev. Genet. 25, 255–271 (2024).

    Article  CAS  PubMed  Google Scholar 

  59. Tarrant, M. K. et al. Regulation of CK2 by phosphorylation and O-GlcNAcylation revealed by semisynthesis. Nat. Chem. Biol. 8, 262–269 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Salguero, A. L. et al. Multifaceted regulation of Akt by diverse C-terminal post-translational modifications. ACS Chem. Biol. 17, 68–76 (2022).

    Article  CAS  PubMed  Google Scholar 

  61. Marotta, N. P. et al. O-GlcNAc modification blocks the aggregation and toxicity of the protein α-synuclein associated with Parkinson’s disease. Nat. Chem. 7, 913–920 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Levine, P. M. et al. α-Synuclein O-GlcNAcylation alters aggregation and toxicity, revealing certain residues as potential inhibitors of Parkinson’s disease. Proc. Natl Acad. Sci. USA 116, 1511–1519 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Balana, A. T. et al. O-GlcNAc forces an α-synuclein amyloid strain with notably diminished seeding and pathology. Nat. Chem. Biol. 20, 646–655 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Fernández-Suárez, M. et al. Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes. Nat. Biotechnol. 25, 1483–1487 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  65. Wu, P. et al. Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag. Proc. Natl Acad. Sci. USA 106, 3000–3005 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Frazier, C. L., Deb, D., Leiter, W. E., Mondal, U. & Weeks, A. M. Raw data and supplementary files for ‘Engineered reactivity of a bacterial E1-like enzyme enables ATP-driven modification of protein C termini’. Dryad https://doi.org/10.5061/dryad.c59zw3rkb (2025).

  67. Hornsby, M. et al. A high through-put platform for recombinant antibodies to folded proteins. Mol. Cell. Proteom. 14, 2833–2847 (2015).

    Article  CAS  Google Scholar 

  68. Zallot, R., Oberg, N. & Gerlt, J. A. The EFI Web Resource for genomic enzymology tools: leveraging protein, genome and metagenome databases to discover novel enzymes and metabolic pathways. Biochemistry 58, 4169–4182 (2019).

    Article  CAS  PubMed  Google Scholar 

  69. Oberg, N., Zallot, R. & Gerlt, J. A. EFI-EST, EFI-GNT and EFI-CGFP: Enzyme Function Initiative (EFI) Web Resource for genomic enzymology tools. J. Mol. Biol. 435, 168018 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Mistry, J. et al. Pfam: the protein families database in 2021. Nucleic Acids Res. 49, D412–D419 (2020).

    Article  PubMed Central  Google Scholar 

  71. UniProt Consortium. UniProt: the Universal Protein Knowledgebase in 2023. Nucleic Acids Res. 51, D523–D531 (2022).

    Article  Google Scholar 

  72. Zheng, J.-S., Tang, S., Qi, Y.-K., Wang, Z.-P. & Liu, L. Chemical synthesis of proteins using peptide hydrazides as thioester surrogates. Nat. Protoc. 8, 2483–2495 (2013).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank S. Coyle, D. Sashital, T. Galateo, R. Rajasekaran, H. Bridge, L. Campbell, L. Mazurkiewicz, E. Johnson and members of the Weeks laboratory for helpful discussions. This work was supported in part by startup funds from the University of Wisconsin – Madison Department of Biochemistry and by an NIH Director’s New Innovator Award (DP2GM149548) to A.M.W. C.L.F. was supported in part by the University of Wisconsin – Madison Biotechnology Training Program under grant number NIH 5 T32 GM135066 and by a William H. Peterson Graduate Fellowship from the University of Wisconsin – Madison Department of Biochemistry. W.E.L. was supported in part by the National Institute of General Medical Sciences of the National Institutes of Health under award number T32 GM152341 (Chemistry–Biology Interface Training Program). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the paper.

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Author notes

  1. These authors contributed equally: Clara L. Frazier, Debashrito Deb.

Authors and Affiliations

  1. Department of Biochemistry, University of Wisconsin – Madison, Madison, WI, USA

    Clara L. Frazier, Debashrito Deb & Amy M. Weeks

  2. Department of Chemistry, University of Wisconsin – Madison, Madison, WI, USA

    William E. Leiter, Umasankar Mondal & Amy M. Weeks

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  1. Clara L. Frazier
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Contributions

C.L.F., D.D., W.E.L., U.M. and A.M.W. designed the experiments. C.L.F., D.D., W.E.L., U.M. and A.M.W. performed experiments. C.L.F., D.D., W.E.L., U.M. and A.M.W. analysed data. C.L.F., D.D. and A.M.W. wrote the paper. C.L.F., D.D., W.E.L., U.M. and A.M.W. reviewed and edited the paper.

Corresponding author

Correspondence to Amy M. Weeks.

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Competing interests

The Wisconsin Alumni Research Foundation has filed a provisional patent application related to this work on which C.L.F., D.D. and A.M.W. are inventors. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 MccB-generated thioesters undergo transthioesterification, S-to-N acyl transfer, and native chemical ligation.

(a) MccA-N7G thioester can undergo transthioesterification with MPAA, a thiol nucleophile that cannot directly capture Mcc-N7G-O-AMP. (b) MccA-N7G-O-AMP can undergo thioesterification and S-to-N acyl shift with Cys to form a peptide bond. (c) In the presence of ATP and Mesna, MccB catalyzes native chemical ligation between an unactivated peptide and an N-terminal Cys peptide.

Extended Data Fig. 2 MccB homologs from L. johnsonii and H. pylori can be used in combination with subtiligase for enzyme-catalysed expressed protein ligation.

(a) LjMccB- and subtiligase-catalysed ATP-dependent peptide ligation of Ala-Phe (left) or AFAGAGS-azAla (right) to GFP- LjTeCH. (b) HpMccB- and subtiligase-catalysed ATP-dependent peptide ligation of Ala-Phe (left) or AFAGAGS-azAla (right) to GFP-HpTeCH.

Extended Data Fig. 3 Comparison of MccB/subtiligase-catalysed and eSrtA-catalysed C-terminal protein modification.

(a) MccB/subtiligase-catalysed C-terminal peptide ligation to GS-GFP-TeCH. In the absence of peptide nucleophile, MccB and subtiligase catalyse GS-GFP cyclization, but this reaction is efficiently suppressed in the presence of 5 mM Ala-Phe. (b) eSrtA-catalysed C-terminal modification of GS-GFP-LPETGG. In the absence of nucleophile, eSrtA catalyzes GFP cyclization that cannot be completely suppressed even in the presence of 10 mM GGG peptide. (c) eSrtA cyclization is suppressed by removing the N-terminal GS sequence at the N terminus of GS-GFP-LPETGG.

Extended Data Fig. 4 Dual N- and C-terminal labeling of GS-MBP-TeCH using eSrtA and MccB/subtiligase.

(a) Scheme for dual N- and C-terminal label of GS-MBP-TeCH with MccB/subtiligase and eSrtA. The magenta circle represent azidoAla and the cyan circle represents 5-FAM. (b) Telescoping one-pot dual labeling of GS-MBP-TeCH with eSrtA and MccB/subtiligase. (c) Concurrent one-pot dual labeling of GS-MBP-TeCH with eSrtA and MccB/subtiligase.

Extended Data Fig. 5 Combining HsMccB-catalysed peptide thioester synthesis with Ubc9-catalysed lysine acylation.

(a) Scheme for lysine acylation using an HsMccB-generated thioester and GFP with an internal minimal LACE tag sequence (IKQE). (b) Scheme for lysine acylation using an HsMccB-generated thioester and GFP with a full length LACE tag sequence (PRKVIKMESEE). (c) Optimization of peptide thioester concentration in LACE reactions. (d) Optimization of thiol concentration at pH 7.6. Excess thiol suppresses the LACE reaction. (e) Optimization of thiol concentration at pH 8.0. Excess thiol suppresses the LACE reaction, which proceeds to higher yield at pH 8.0 compared to 7.6.

Supplementary information

Supplementary Information

Supplementary Tables 1–5 and Figs. 1–84.

Source data

Source Data Fig. 1

Unprocessed kinetics and conversion data for MccB-catalysed peptide reactions.

Source Data Fig. 2

Unprocessed extracted ion chromatograms and spectra for MccB-catalysed peptide reactions with nucleophile.

Source Data Fig. 3

Unprocessed deconvoluted mass spectra for MccB-catalysed thioesterification and modification of TeCH-tagged proteins.

Source Data Fig. 4

Heatmap intensity values and deconvoluted mass spectra for MccB homologue-catalysed protein modification.

Source Data Fig. 5

Unprocessed deconvoluted mass spectra for MccB application to enzyme-catalysed expressed protein ligation; unprocessed TIFF files from fluorescence microscopy experiments.

Source Data Fig. 6

Heatmap intensity values and unprocessed deconvoluted mass spectra for MccB application to the LACE system.

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Frazier, C.L., Deb, D., Leiter, W.E. et al. Engineered reactivity of a bacterial E1-like enzyme enables ATP-driven modification of protein and peptide C termini. Nat. Chem. 17, 1371–1382 (2025). https://doi.org/10.1038/s41557-025-01871-3

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