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A host dTMP-bound structure of T4 phage dCMP hydroxymethylase mutant using an X-ray free electron laser
摘要： 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.
关键词： hydroxymethylation,XFEL,dCMP hydroxymethylase,X-ray free electron laser,T4dCH,hydroxylation,cytosine bases,phage DNA protection,dTMP,Escherichia coli,bacteriophage T4,methylene intermediate
更新于2025-09-12 10:27:22
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Photoelectrochemical biosensor for hydroxymethylated DNA detection and T4-β-glucosyltransferase activity assay based on WS2 nanosheets and carbon dots
摘要： 5-Hydroxymethylcytosine (5hmC) plays an important role in switching genes on and off in mammals, and it is implicated in both embryonic development and cancer progression. Herein, a novel photoelectrochemical (PEC) biosensor was developed for 5hmC detection based on WS2 nanosheets as the photoactive material and boronic acid functionalized carbon dots (B-CDs) for signal amplification unit. This biosensor can also be used for T4-β-glucosyltransferase (β-GT) activity assessment. Firstly, WS2 nanosheets and gold nanoparticles (AuNPs) were immobilized on an ITO electrode surface. Then probe DNA was immobilized on this electrode surface via Au-S bond. Afterwards, the complementary DNA containing 5hmC was then captured on the modified electrode surface by hybridization. Subsequently, β-GT transferred glucose from uridine diphosphoglucose to the hydroxyl groups of the 5hmC residues. After glycosylation, B-CDs could further be immobilized on the modified electrode surface resulting in a strong photocurrent. The PEC biosensor afforded high selectivity, excellent sensitivity and good reproducibility, with detection limits of 0.0034 nM and 0.028 unit/mL for 5hmC and β-GT, respectively. Results demonstrate that the photoelectrochemical strategy introduced here based on WS2 nanosheets and B-CDs offers a versatile platform for hydroxymethylated DNA detection, β-GT activity assessment and β-GT inhibitor screening.
关键词： Photoelectrochemical biosensor,DNA hydroxymethylation,β-Glucosyltransferase,WS2 nanosheets,carbon dots
更新于2025-09-09 09:28:46