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. Author manuscript; available in PMC: 2026 Jul 18.
Published in final edited form as: Nat Chem. 2025 Jul 18;17(9):1371–1382. doi: 10.1038/s41557-025-01871-3

Figure 6. Enzymatic synthesis of ubiquitin-derived peptide thioesters using an MccB homolog with relaxed substrate specificity.

Figure 6.

(A) Synthetic ubiquitin-derived peptide C-terminal thioesters are substrates for Ubc9 in the lysine acylation with conjugating enzymes (LACE) strategy. (B) MccB-catalyzed synthesis of peptide C-terminal thioesters. (C) MccB from Histophilus somni (cyan, HsMccB) acts on a basic substrate and exhibited lower substrate specificity in our screen of MccB homologs. (D) HsMccB efficiently converts Ubc9 substrate peptides for LACE to Mes thioesters (left), while AcCysNHMe (right) was a less effective thiol donor. (E) Application of MccB-generated thioesters for acylation of an internal lysine side chain in LACE-tagged GFP. Left, acylation using 300 μM LRLRGG-Mes thioester. Right, acylation using 750 μM MccB-generated MLGLRGG-Mes thioester.