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.
Highlights
The X-ray free electron laser (XFEL) is a new fourth-generation light source that produces considerably bright X-ray pulses[1,2,3,4]
After the XFEL structures of photosystem I (PDB ID: 3PCQ)[13] and the photosynthetic reaction center (PDB ID: 4AC5)[14] were solved with the Linac Coherent Light Source (LCLS), many structures including G protein-coupled receptors for drug design and photoreactive proteins for molecular motion were determined using XFEL data collected at XFEL facilities[8,15,16,17,18,19,20,21]
The studied proteins are largely limited to some classes of proteins such as photoactivating proteins for time-resolved (TR) experiments, membrane proteins that are stable in the lipidic cubic phase (LCP), and robust model proteins[23,24]
Summary
The X-ray free electron laser (XFEL) is a new fourth-generation light source that produces considerably bright X-ray pulses[1,2,3,4]. The overall model of T4dCH D179N fitted in well with an electron density map generated by XFEL (Fig. 2). We carefully explored the XFEL structure and found that this electron density matches with the sodium ion that forms an octahedral geometry with the main chain oxygen atoms of Lys[72], Ile[74], and Gly[76], and side chain hydroxyl group of Thr[78], in addition to two water molecules (Supplementary Fig. S3b).
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