Abstract
The biosynthesis of ribosomally synthesized and posttranslationally modified peptides (RiPPs) leverages iterative catalysis to enhance structural and biological diversity. Traditionally, iterative enzymes install posttranslational modifications on linear peptides, rather than mature RiPPs with intricate three-dimensional structures, which require complex changes in substrate binding. Here we present a prolific class of GCN5-related N-acetyltransferases (GNATs) that iteratively and consecutively acylate two Lys residues within the loop and ring motifs of lasso peptides, diverging from the typical iterative modification of linear peptides. Utilizing high-resolution cryogenic-electron microscopy and enzymatic reconstitution, we define the lasso peptide-binding pocket of IatT and pinpoint key residues that distinguish its two distinct acetylation steps. Structure-based engineering of IatT’s acetyl-recognition site expands the cavity to accommodate longer-chain acyl groups, enabling the creation of lipolasso peptides, a class of ribosomal lipopeptide. This engineering strategy can be applied to any RiPP biosynthetic gene cluster encoding GNAT, facilitating the efficient diversification of ribosomal lipopeptides.
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Data availability
The data that support the findings of this study are available within the main text and Supplementary Information. The cryo-EM maps of IatT in complex with CoA, AcCoA and AcCoA-2 have been deposited into the Electron Microscopy Database with accession codes EMD-60983, EMD-60984 and EMD-64004, respectively. The corresponding coordinates have been deposited into the PDB with accession codes 9IY3, 9IY4 and 9UBC, respectively. In addition, D7 symmetry-averaged sharpened and unsharpened maps for the IatT-AcCoA-2 whole particle are deposited as additional maps under the corresponding accession code. AMBER parameters were obtained from an open-source database available at http://pc164.materials.uoi.gr/dpapageo/amberparams.php. Data are available from the corresponding authors upon request. Source data are provided with this paper.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (grant nos. 22077056 to S.-H.D., 22477050 to S.L., 32171300 to D.L., 22107040 to J.-J.C., 22377046 and 21907046 to S.-H.D., and 12374011 to Y.P.), National Key Research and Development Program of China (2022YFC2903504 to Y.P.), and The Science and Technology Major Program of Gansu Province of China (grant nos. 22ZD6FA006, 23ZDFA015 and 24ZD13FA017 to S.-H.D.).
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J.X. and S.-H.D. carried out bioinformatic, genetic and biochemical work and performed metabolic analysis, compound isolation and structure elucidation. S.W., Y.P. and D.L. carried out cryo-EM data collection and structure elucidation. Z.-Q.L. started the project and performed heterologous expression and isolation of mirusins. X.-T.G., S.F., F.-Y.T. and J.-J.C. participated in the heterologous expression and cloning experiments. Q.W. facilitated in MALDI–TOF MS data collection. X.W. and K.K.H. performed the native mass data collection and analysis. K.G. supervised and provided guidance for the project. S.L. and S.-H.D. designed, conceived and supervised the project. S.L., D.L. and S.-H.D. analyzed data and wrote the paper with input from all authors.
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Extended data
Extended Data Fig. 1 The bioinformatic analysis of RiPP-associated GNATs.
a, The IatT-containing SSN cluster with 40% (left) and 70% (right) sequence identity threshold. The green and red nodes represent the GNAT sequences of class I and II, respectively. All nodes are nonredundant with identical sequences displayed as a single node. b, The ML tree of RiPP-associated GNATs. The characterized GNATs involved in ribosomal lipopeptide biosynthesis are represented by solid green circles. Class II lasso peptide GNATs are marked with solid red circles, whereas the two exceptions with a Lys5 instead of Lys4 are denoted by purple circles. All other GNATs belong to class I lasso peptides. Characterized lasso peptide GNATs are highlighted in bold blue names, while other IatT-like GNATs are indicated by their UniProt IDs.
Extended Data Fig. 2 Heterologous expression of ven BGC.
a,b, HRMS data for heterologous expression products of ven BGC (a) and ven BGC with VenA-G4K mutation (b). The observed and calculated masses ([M + 2H]2+) of each product are displayed on the corresponding spectra. The C-terminal Cys residue of VenA highlighted by a green dashed rectangle is not present in 4-6. All assays were run in triplicate and representative results are shown.
Extended Data Fig. 3 Binding analysis of IatA and 2* with IatT.
a,b, Structural comparison of IatT-IatA AlphaFold-multimer model with SpeG in complex with spermine related products (a) and PseH bound with AcCoA (b). c, ITC data for titration of 2* into IatT solution. No obvious binding is observed. All assays were run in triplicate and representative results are shown.
Extended Data Fig. 4 Enzymatic activity assays of IatT and homologous GNATs in vitro via LC-HRMS analysis.
a, EICs for assays of IatT using 2* and 1* as substrates. b-h, EICs for assays of AlbT (b), VenT (c), EmbT (d), NocT (e), XiaT (f), JiaT (g), and AlkT (h) using 1 and 2 as substrates. All assays were run in triplicate and representative results are shown.
Extended Data Fig. 5 Flow-chart for cryo-EM data processing.
Shown the data processing procedures for entire protein complex of IatT-CoA (left), IatT-AcCoA (middle), and IatT-AcCoA-2 (right). Details can be found in the Methods section.
Extended Data Fig. 6 Resolution analysis of cryo-EM 3D reconstruction.
Top, overall density maps of the final 3D reconstruction; bottom, the gold-standard Fourier shell correlation (FSC) curves for the 3D reconstruction calculated in cryoSPARC. FSC = 0.143 is indicated.
Extended Data Fig. 7 Conformations 1a (a), 1b (b), 1c (c), 1 d (d), and 1e (e) of lasso peptide 1 in the IatT pocket.
Extended Data Fig. 8 Conformations 1 f (a), 1 g (b), and 1 h (c) of lasso peptide 1 and a different view of the conformation of lasso peptide 2 (d) in the IatT pocket.
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Supplementary Figs. 1–39 and Tables 1–7.
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Unprocessed SDS–PAGE gels.
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DNA sequences.
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Computational conformation analysis.
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Computational conformation analysis.
Source Data Extended Data Fig. 8 (download ZIP )
Computational conformation analysis.
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Xiong, J., Wu, S., Liang, ZQ. et al. Iterative acylation on mature lasso peptides by widespread acetyltransferases. Nat Chem Biol (2026). https://doi.org/10.1038/s41589-026-02149-6
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DOI: https://doi.org/10.1038/s41589-026-02149-6