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 journalArticle

Abstract

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
Volume9
Issue number1
DOIs
Publication statusPublished - 2019 Dec 1

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Bacteriophage T4
Lasers
X-Rays
Electrons
Catalytic Domain
Escherichia coli
Cytosine
Hydroxylation
Bacteriophages
Temperature
Water
DNA

ASJC Scopus subject areas

  • General

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A host dTMP-bound structure of T4 phage dCMP hydroxymethylase mutant using an X-ray free electron laser. / Park, Si Hoon; Park, Jaehyun; Lee, Sang Jae; Yang, Woo Seok; Park, Sehan; Kim, Kyungdo; Park, Zee Yong; Song, Hyun Kyu.

In: Scientific reports, Vol. 9, No. 1, 16316, 01.12.2019.

Research output: Contribution to journalArticle

Park, Si Hoon ; Park, Jaehyun ; Lee, Sang Jae ; Yang, Woo Seok ; Park, Sehan ; Kim, Kyungdo ; Park, Zee Yong ; Song, Hyun Kyu. / A host dTMP-bound structure of T4 phage dCMP hydroxymethylase mutant using an X-ray free electron laser. In: Scientific reports. 2019 ; Vol. 9, No. 1.
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