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

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...

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Detalles Bibliográficos
Autores principales: Park, Si Hoon, Park, Jaehyun, Lee, Sang Jae, Yang, Woo Seok, Park, Sehan, Kim, Kyungdo, Park, Zee-Yong, Song, Hyun Kyu
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6841964/
https://www.ncbi.nlm.nih.gov/pubmed/31705139
http://dx.doi.org/10.1038/s41598-019-52825-y
Descripción
Sumario: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.