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Analysis of RNA Methylation by Phylogenetically Diverse Cfr Radical S-Adenosylmethionine Enzymes Reveals an Iron-Binding Accessory Domain in a Clostridial Enzyme

  • James D. Gumkowski
    James D. Gumkowski
    Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, United States
  • Ryan J. Martinie
    Ryan J. Martinie
    Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, United States
  • Patrick S. Corrigan
    Patrick S. Corrigan
    Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, United States
  • Juan Pan
    Juan Pan
    Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, United States
    More by Juan Pan
  • Matthew R. Bauerle
    Matthew R. Bauerle
    Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, United States
  • Mohamed Almarei
    Mohamed Almarei
    Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, United States
  • Squire J. Booker
    Squire J. Booker
    Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, United States
    Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, United States
    Howard Hughes Medical Institute, The Pennsylvania State University, University Park, Pennsylvania 16802, United States
  • Alexey Silakov
    Alexey Silakov
    Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, United States
  • Carsten Krebs*
    Carsten Krebs
    Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, United States
    Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, United States
    *E-mail: [email protected]
  • , and 
  • Amie K. Boal*
    Amie K. Boal
    Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, United States
    Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, United States
    *E-mail: [email protected]
    More by Amie K. Boal
Cite this: Biochemistry 2019, 58, 29, 3169–3184
Publication Date (Web):June 27, 2019
https://doi.org/10.1021/acs.biochem.9b00197
Copyright © 2019 American Chemical Society

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    Abstract

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    Cfr is a radical S-adenosylmethionine (SAM) RNA methylase linked to multidrug antibiotic resistance in bacterial pathogens. It catalyzes a chemically challenging C–C bond-forming reaction to methylate C8 of A2503 (Escherichia coli numbering) of 23S rRNA during ribosome assembly. The cfr gene has been identified as a mobile genetic element in diverse bacteria and in the genome of select Bacillales and Clostridiales species. Despite the importance of Cfr, few representatives have been purified and characterized in vitro. Here we show that Cfr homologues from Bacillus amyloliquefaciens, Enterococcus faecalis, Paenibacillus lautus, and Clostridioides difficile act as C8 adenine RNA methylases in biochemical assays. C. difficile Cfr contains an additional Cys-rich C-terminal domain that binds a mononuclear Fe2+ ion in a rubredoxin-type Cys4 motif. The C-terminal domain can be truncated with minimal impact on C. difficile Cfr activity, but the rate of turnover is decreased upon disruption of the Fe2+-binding site by Zn2+ substitution or ligand mutation. These findings indicate an important purpose for the observed C-terminal iron in the native fusion protein. Bioinformatic analysis of the C. difficile Cfr Cys-rich domain shows that it is widespread (∼1400 homologues) as a stand-alone gene in pathogenic or commensal Bacilli and Clostridia, with >10% encoded adjacent to a predicted radical SAM RNA methylase. Although the domain is not essential for in vitro C. difficile Cfr activity, the genomic co-occurrence and high abundance in the human microbiome suggest a possible functional role for a specialized rubredoxin in certain radical SAM RNA methylases that are relevant to human health.

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    Supporting Information

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.9b00197.

    • Tables S1–S4 and Figures S1–S9 (PDF)

    Accession Codes

    WP_002578771, C. difficile strain QCD63q42; WP_015735625, P. lautus sp. Y412MC10; WP_013351126, B. amyloliquefaciens strain DSM-7 (ATCC 23350); WP_002405682, E. faecalis strain TX0635; AIT41446, S. aureus (plasmid); WP_003483120, C. sporogenes strain ATCC 15579.

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    Cited By

    This article is cited by 2 publications.

    1. Nozomu Obana, Hiraku Takada, Caillan Crowe-McAuliffe, Mizuki Iwamoto, Artyom A Egorov, Kelvin J Y Wu, Shinobu Chiba, Victoriia Murina, Helge Paternoga, Ben I C Tresco, Nobuhiko Nomura, Andrew G Myers, Gemma C Atkinson, Daniel N Wilson, Vasili Hauryliuk. Genome-encoded ABCF factors implicated in intrinsic antibiotic resistance in Gram-positive bacteria: VmlR2, Ard1 and CplR. Nucleic Acids Research 2023, 51 (9) , 4536-4554. https://doi.org/10.1093/nar/gkad193
    2. Joshua T. McLean, Alby Benny, Mark D. Nolan, Glenna Swinand, Eoin M. Scanlan. Cysteinyl radicals in chemical synthesis and in nature. Chemical Society Reviews 2021, 50 (19) , 10857-10894. https://doi.org/10.1039/D1CS00254F

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