Dataset Information

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

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.

SUBMITTER: Park SH

PROVIDER: S-EPMC6841964 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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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 SH Park Jaehyun J Lee Sang Jae SJ Yang Woo Seok WS Park Sehan S Kim Kyungdo K Park Zee-Yong ZY Song Hyun Kyu HK

Scientific reports 20191108 1

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 ha ...[more]

PMID: 31705139

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Project description:The fate of biomolecules in the environment depends in part on understanding the surface chemistry occurring at the biological-geochemical (bio-geo) interface. Little is known about how environmental DNA (eDNA) or smaller components, like nucleotides and oligonucleotides, persist in aquatic environments and the role of surface interactions. This study aims to probe surface interactions and adsorption behavior of nucleotides on oxide surfaces. We have investigated the interactions of individual nucleotides (dGMP, dCMP, dAMP, and dTMP) on TiO2 particle surfaces as a function of pH and in the presence of complementary and noncomplementary base pairs. Using attenuated total reflectance-Fourier transform infrared spectroscopy, there is an increased number of adsorbed nucleotides at lower pH with a preferential interaction of the phosphate group with the oxide surface. Additionally, differential adsorption behavior is seen where purine nucleotides are preferentially adsorbed, with higher surface saturation coverage, over their pyrimidine derivatives. These differences may be a result of intermolecular interactions between coadsorbed nucleotides. When the TiO2 surface was exposed to two-component solutions of nucleotides, there was preferential adsorption of dGMP compared to dCMP and dTMP, and dAMP compared to dTMP and dCMP. Complementary nucleotide base pairs showed hydrogen-bond interactions between a strongly adsorbed purine nucleotide layer and a weaker interacting hydrogen-bonded pyrimidine second layer. Noncomplementary base pairs did not form a second layer. These results highlight several important findings: (i) there is differential adsorption of nucleotides; (ii) complementary coadsorbed nucleotides show base pairing with a second layer, and the stability depends on the strength of the hydrogen bonding interactions and; (iii) the first layer coverage strongly depends on pH. Overall, the importance of surface interactions in the adsorption of nucleotides and the templating of specific interactions between nucleotides are discussed.

Project description:The use of phages as antibacterial agents is limited by their generally narrow host ranges. The aim of this study was to make a T4-like phage, WG01, obtain the host range of another T4-like phage, QL01, by replacing its host-determinant gene region with that of QL01. This process triggered a direct expansion of the WG01 host range. The offspring of WG01 obtained the host ranges of both QL01 and WG01, as well as the ability to infect eight additional host bacteria in comparison to the wild-type strains. WQD had the widest host range; therefore, the corresponding fragments, named QD, could be used for constructing a homologous sequence library. Moreover, after a sequencing analysis of gene 37, we identified two different mechanisms responsible for the expanded host range: (i) the first generation of WG01 formed chimeras without mutations, and (ii) the second generation of WG01 mutants formed from the chimeras. The expansion of the host range indicated that regions other than the C-terminal region may indirectly change the receptor specificity by altering the supportive capacity of the binding site. Additionally, we also found the novel means by which subsequent generations expanded their host ranges, namely, by exchanging gene 37 to acquire a wider temperature range for lysis. The method developed in this work offers a quick way to change or expand the host range of a phage. Future clinical applications for screening phages against a given clinical isolate could be achieved after acquiring more suitable homologous sequences.IMPORTANCE T4-like phages have been established as safe in numerous phage therapy applications. The primary drawbacks to the use of phages as therapeutic agents include their highly specific host ranges. Thus, changing or expanding the host range of T4-like phages is beneficial for selecting phages for phage therapy. In this study, the host range of the T4-like phage WG01 was expanded using genetic manipulation. The WG01 derivatives acquired a novel means of expanding their host ranges by acquiring a wider temperature range for lysis. A region was located that had the potential to be used as a sequence region for homologous sequence recombination.

Dataset Information

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

Publications

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

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