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Characterization of a Unique Interrupted Adenylation Domain That Can Catalyze Three Reactions

  • Taylor A. Lundy
    Taylor A. Lundy
    Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536-0596, United States
    More by Taylor A. Lundy
  • , 
  • Shogo Mori
    Shogo Mori
    Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536-0596, United States
    More by Shogo Mori
  • , 
  • Nishad Thamban Chandrika
    Nishad Thamban Chandrika
    Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536-0596, United States
    More by Nishad Thamban Chandrika
    Orcidhttp://orcid.org/0000-0002-3605-169X
  • , and 
  • Sylvie Garneau-Tsodikova*
    Sylvie Garneau-Tsodikova
    Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536-0596, United States
    *E-mail: [email protected]
    More by Sylvie Garneau-Tsodikova
    Orcidhttp://orcid.org/0000-0002-7961-5555
Cite this: ACS Chem. Biol. 2020, 15, 1, 282–289
Publication Date (Web):December 30, 2019
https://doi.org/10.1021/acschembio.9b00929
Copyright © 2019 American Chemical Society
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    Abstract

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    Interrupted adenylation (A) domains contain auxiliary domains within their structure and are a subject of growing interest in the field of nonribosomal peptide biosynthesis. They have been shown to possess intriguing functions and structure as well as promising engineering potential. Here, we present the characterization of an unprecedented type of interrupted A domain from the columbamides biosynthetic pathway, ColG(AMsMbA). This interrupted A domain contains two back-to-back methylation (M) domains within the same interruption site in the A domain, whereas previously, naturally occurring reported and characterized interrupted A domains harbored only one M domain. By a series of radiometric and mass spectrometry assays, we show that the first and second M domains site specifically methylate the side-chain oxygen and backbone nitrogen of l-Ser after the substrate is transferred onto a carrier thiolation domain, ColG(T). This is the first reported characterization of a dimethylating back-to-back interrupted A domain. The insights gained by this work lay the foundation for future combinatorial biosynthesis of site specifically methylated nonribosomal peptides.

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschembio.9b00929.

    • Experimental procedures for all molecular cloning, overexpression, and purification of proteins used for this study, all biochemical assays, and chemical synthesis. Tables of primers used and expression constructs made (Tables S1–S3). Figures showing SDS-PAGE gels (Figures S1 and S3), sequence alignments (Figure S2), apo to holo conversion of ColG(T) (Figure S4), synthetic schemes and NMR spectra for N,O-diMe-l-Ser (Figures S5–S11), and the major MS/MS fragment masses of the standards l-Ser and its methylated derivatives (Figure S12) (PDF)

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    Cited By

    This article is cited by 7 publications.

    1. Fumitaka Kudo, Takuji Chikuma, Mizuki Nambu, Taichi Chisuga, Shimpei Sumimoto, Arihiro Iwasaki, Kiyotake Suenaga, Akimasa Miyanaga, Tadashi Eguchi. Unique Initiation and Termination Mechanisms Involved in the Biosynthesis of a Hybrid Polyketide-Nonribosomal Peptide Lyngbyapeptin B Produced by the Marine Cyanobacterium Moorena bouillonii. ACS Chemical Biology 2023, 18 (4) , 875-883. https://doi.org/10.1021/acschembio.3c00011
    2. Gina Porras, François Chassagne, James T. Lyles, Lewis Marquez, Micah Dettweiler, Akram M. Salam, Tharanga Samarakoon, Sarah Shabih, Darya Raschid Farrokhi, Cassandra L. Quave. Ethnobotany and the Role of Plant Natural Products in Antibiotic Drug Discovery. Chemical Reviews 2021, 121 (6) , 3495-3560. https://doi.org/10.1021/acs.chemrev.0c00922
    3. Milda Kaniusaite, Tiia Kittilä, Robert J. A. Goode, Ralf B. Schittenhelm, Max J. Cryle. Redesign of Substrate Selection in Glycopeptide Antibiotic Biosynthesis Enables Effective Formation of Alternate Peptide Backbones. ACS Chemical Biology 2020, 15 (9) , 2444-2455. https://doi.org/10.1021/acschembio.0c00435
    4. Evan T. Miller, Oleg V. Tsodikov, Sylvie Garneau-Tsodikova. Structural insights into the diverse prenylating capabilities of DMATS prenyltransferases. Natural Product Reports 2024, 41 (1) , 113-147. https://doi.org/10.1039/D3NP00036B
    5. Taylor A. Lundy, Shogo Mori, Sylvie Garneau-Tsodikova. A thorough analysis and categorization of bacterial interrupted adenylation domains, including previously unidentified families. RSC Chemical Biology 2020, 1 (4) , 233-250. https://doi.org/10.1039/D0CB00092B
    6. Shogo Mori, Sylvie Garneau-Tsodikova, Oleg V. Tsodikov. Unimodular Methylation by Adenylation–Thiolation Domains Containing an Embedded Methyltransferase. Journal of Molecular Biology 2020, 432 (21) , 5802-5808. https://doi.org/10.1016/j.jmb.2020.09.004
    7. Taylor A. Lundy, Shogo Mori, Sylvie Garneau-Tsodikova. Lessons learned in engineering interrupted adenylation domains when attempting to create trifunctional enzymes from three independent monofunctional ones. RSC Advances 2020, 10 (56) , 34299-34307. https://doi.org/10.1039/D0RA05490A
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