A host dTMP-bound structure of T4 phage dCMP hydroxymethylase mutant using an X-ray free electron laser

Si Hoon Park, Jaehyun Park, Sang Jae Lee, Woo Seok Yang, Sehan Park, Kyungdo Kim, Zee Yong Park, Hyun Kyu Song

Research output: Contribution to journalArticlepeer-review

3 Citations (Scopus)


The hydroxymethylation of cytosine bases plays a vital role in the phage DNA protection system inside the host Escherichia coli. This modification is known to be catalyzed by the dCMP hydroxymethylase from bacteriophage T4 (T4dCH); structural information on the complexes with the substrate, dCMP and the co-factor, tetrahydrofolate is currently available. However, the detailed mechanism has not been understood clearly owing to a lack of structure in the complex with a reaction intermediate. We have applied the X-ray free electron laser (XFEL) technique to determine a high-resolution structure of a T4dCH D179N active site mutant. The XFEL structure was determined at room temperature and exhibited several unique features in comparison with previously determined structures. Unexpectedly, we observed a bulky electron density at the active site of the mutant that originated from the physiological host (i.e., E. coli). Mass-spectrometric analysis and a cautious interpretation of an electron density map indicated that it was a dTMP molecule. The bound dTMP mimicked the methylene intermediate from dCMP to 5′-hydroxymethy-dCMP, and a critical water molecule for the final hydroxylation was convincingly identified. Therefore, this study provides information that contributes to the understanding of hydroxymethylation.

Original languageEnglish
Article number16316
JournalScientific reports
Issue number1
Publication statusPublished - 2019 Dec 1

Bibliographical note

Funding Information:
We would like to thank all the members of PAL-XFEL. This study was supported by a National Research Foundation of Korea (NRF) grant from the Korean government (Grant No. 2016R1E1A1A01942623).

Publisher Copyright:
© 2019, The Author(s).

ASJC Scopus subject areas

  • General


Dive into the research topics of 'A host dTMP-bound structure of T4 phage dCMP hydroxymethylase mutant using an X-ray free electron laser'. Together they form a unique fingerprint.

Cite this