Abstract
Nucleic acid modifying enzymes drive diverse defense and counter-defense measures in the evolutionary arms race between viruses and their cellular hosts. Abundant and widespread bacterial viruses (bacteriophage or phage) encode for biosynthetic pathways that install elaborate DNA hypermodifications which protect their genomic DNA from host endonucleases. Here, we establish the molecular basis for the multistep biosynthesis of 6-aminocarboxymethyl-2ʹ-deoxyadenosine (6-NcmdA), a nucleobase hypermodification found in the virion DNA of bacteriophage Mu that leads to restriction evasion in the context of phage-host conflicts. In the first step, we show that Mu-encoded Mom enzyme catalyzes the formation of 6-NcmdA by transferring glycine from charged tRNAGly to the N6 position of adenine within double-stranded DNA. We uncover a second step where the glycyl-dA intermediate undergoes an on-base rearrangement to form 6-NcmdA. Examination of the proposed reaction pathways by quantum chemical calculations confirms the instability of acyl exocyclic groups at N6-adenine and reveals an energetically favorable orientation of 6-NcmdA that restores canonical base pairing. An X-ray structure confirms Mom is a member of the GNAT superfamily and suggests binding sites for both tRNA and DNA. Guided by the Mom structure and patterns of sequence conservation across metagenomic space, we show residues R111 and S124 are essential for catalysis. This work demonstrates that the Mom enzyme defines a new category of acetyltransferases utilizing charged tRNA to modify DNA.
Data availability
The final model of the structure solved in this work was deposited in the Protein Data Bank under accession code 8BV8. ORCA output files for all energy minimizations of educts, products, and the spirocyclic intermediate, as well as ORCA output files for the saddle points/transition states, and the corresponding PDB files (translated from the ORCA files using Avogadro) are made available in a Zip drive. A sequencing read archive (SRA) containing BAM files produced by the PacBio SMRT sequencing platform and extended to include kinetic data for the modification motif analysis in this work can be obtained at NCBI Bioproject PRJNA1406289. Metagenome information for Mom Homolog A can be found at https://img.jgi.doe.gov/cgi-bin/m/main.cgi?section=TaxonDetail&page=taxonDetail&taxon_oid=3300002484. Metagenome information for Mom Homolog B can be found at https://img.jgi.doe.gov/cgi-bin/m/main.cgi?section=TaxonDetail&page=taxonDetail&taxon_oid=2872672955. All other data supporting the findings of this work are available within the Article, Supplementary Information, or from the corresponding author(s) upon request. Source data are provided with this paper.
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Acknowledgements
We thank Johanna Hakanpää and Guillaume Pompidor for access to the PETRA III P11 beamline at DESY, and Dr. Honorata Czapińska, Dr. Elzbieta Nowak, and Michal Pastor for data collection. We gratefully acknowledge Poland's high-performance Infrastructure PLGrid [ACK Cyfronet AGH] for providing computer facilities and support within computational grant no [plgmomylation and plgmomylation2]. Core facility support at IIMCB was aided by IN-MOL-CELL Infrastructure (RRID:SCR_021630) funded by the EU NextGenerationEU under National Recovery and Resilience Plan Horizon Europe (Project 101059801 - RACE) and European Funds for Smart Economy 2021-2027 (FENG) RACE-PRIME project within the International Research Agenda Program Fundacja na rzecz Nauki Polskiej (Foundation for Polish Science) program. The authors are grateful for purified WT GlyRS and WT AlaRS from Corinna Tuckey, Dr. Ying Zhou, Dr. Bradley J. Landgraf, and Dr. Emily Chen, T7 RNA polymerase from Professor Janusz Bujnicki’s lab, a plasmid encoding tRNAGly/GCC from Dr. Haruichi Asahara, and plasmid pRY from Dr. Robert M. Yarrington. We thank Dr. Nan Dai, Dr. Eric J. Wolf, Dr. Edwin E. Escobar, and the Protein Research Mass Spec Facility at NEB for their expertise using intact mass spectrometry to characterize tRNA. We also thank Dr. Andrew P. Sikkema for sharing his methods and expertise regarding WT CCA2, as well as Dr. Esta Tamanaha and James Elliot for sharing immobilized aptamers during purification of CCA2. Critical readings of our manuscript were provided by Dr. Ivan R. Corrêa Jr., Dr. Richard J. Roberts, Dr. Timothy R. Blower, Dr. Gregory J. S. Lohman, and colleagues at NEB/IIMCB. Tashe José provided key feedback on scientific illustrations. We are thankful to Dr. Shweta Karambelkar for sharing her unpublished thesis from the Indian Institute of Science, Bangalore. R.M.B.S., Y.J.L., C.G., E.A.S., S.R.L., and P.R.W. are grateful for the generous support from the Comb Family and New England Biolabs, Inc. without whom this work would not have been possible. M.B. and A.S. are grateful for funding from the Polish National Science Centre (NCN, 2018/30/Q/NZ2/00669) and the Foundation for Polish Science (FNP, POIR.04.04.00-00-5D81/17-00 to M.B. and START 79.2020 to A.S.). Some figures were made using BioRender. We dedicate this work to the memory of Professor Stanley Hattman.
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P.R.W. and M.B. conceptualized the project. R.M.B.S., A.S., and Y.J.L. developed the methodologies used in this study. R.M.B.S., A.S., Y.J.L., C.G., S.R.L., E.A.R., K.S., M.S.K., M.B., and P.R.W. conducted experiments and completed data analysis. R.M.B.S. and A.S. administered the project and managed data visualization. M.B. and P.R.W. supervised the project and secured funding. R.M.B.S., P.R.W., Y.-J.L., A.S., and M.B. wrote the manuscript with support from all the other authors.
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R.M.B.S., Y.-J.L., C.G., E.A.S., S.R.L., M.S.K., and P.R.W. are employees of New England Biolabs, a manufacturer and vendor of molecular biology reagents. This affiliation does not affect the authors’ impartiality, adherence to journal standards and policies, or availability of data. The remaining authors declare no competing interests.
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Silva, R.M.B., Slyvka, A., Lee, YJ. et al. A single viral enzyme drives tRNA-dependent hypermodification of DNA at adenine. Nat Commun (2026). https://doi.org/10.1038/s41467-026-70671-1
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DOI: https://doi.org/10.1038/s41467-026-70671-1
