A host dTMP-bound structure of T4 phage dCMP hydroxymethylase mutant using an X-ray free electron laser
- Authors
- Park, Si Hoon; Park, Jaehyun; Lee, Sang Jae; Yang, Woo Seok; Park, Sehan; Kim, Kyungdo; Park, Zee-Yong; Song, Hyun Kyu
- Issue Date
- 8-11월-2019
- Publisher
- NATURE PUBLISHING GROUP
- Citation
- SCIENTIFIC REPORTS, v.9
- Indexed
- SCIE
SCOPUS
- Journal Title
- SCIENTIFIC REPORTS
- Volume
- 9
- URI
- https://scholar.korea.ac.kr/handle/2021.sw.korea/61914
- DOI
- 10.1038/s41598-019-52825-y
- ISSN
- 2045-2322
- 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.
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