Structural and biochemical characterization of a nucleotide hydrolase from Streptococcus pneumonia.

In this report, we structurally and biochemically characterized the unknown gene product SP1746 from Streptococcus pneumoniae serotype 4. Various crystal structures of SP1746 in the apo form and in complex with different nucleotides were determined. SP1746 is a globular protein, which belongs to the histidine-aspartate (HD) domain superfamily with two Fe3+ ions in the active site that are coordinated by key active site residues and water molecules. All nucleotides bind in a similar orientation in the active site with their phosphate groups anchored to the diiron cluster. Biochemically, SP1746 hydrolyzes different nucleotide substrates. SP1746 most effectively hydrolyzes diadenosine tetraphosphate (Ap4A) to two ADPs. Based on the aforementioned data, we annotated SP1746 as an Ap4A hydrolase, belonging to the YqeK family. Our in vitro data indicate a potential role for SP1746 in regulating Ap4A homeostasis, which requires validation with in vivo experiments in bacteria in the future.

Crystal structure of the complex of DNA with the C-terminal domain of TYE7 from Saccharomyces cerevisiae.

TYE7, a bHLH (basic helix-loop-helix) transcription factor from Saccharomyces cerevisiae, is involved in the regulation of many genes, including glycolytic genes. Meanwhile, accumulating evidence indicates that TYE7 also functions as a cyclin and is linked to sulfur metabolism. Here, the structure of TYE7 (residues 165-291) complexed with its specific DNA was determined by X-ray crystallography. Structural analysis and comparison revealed that His185 and Glu189 are conserved in base recognition. However, Arg193 is also involved in base recognition in the structures that were compared. In the structure in this study, Arg193 in chain A has two conformations and makes a salt bridge with the phosphate backbone structure. In addition, a series of corresponding electrophoretic mobility shift assays were performed to better understand the DNA-binding mechanism of the bHLH domain of TYE7.

Crystallographic Analysis of the Catalytic Mechanism of Phosphopantothenoylcysteine Synthetase from Saccharomyces cerevisiae.

Phosphopantothenoylcysteine (PPC) synthetase (PPCS) catalyzes nucleoside triphosphate-dependent condensation reaction between 4'-phosphopantothenate (PPA) and l-cysteine to form PPC in CoA biosynthesis. The catalytic mechanism of PPCS has not been resolved yet. Coenzyme A biosynthesis protein 2 (Cab2) possesses activity of PPCS in Saccharomyces cerevisiae. Our enzymatic assays suggest that Cab2 could utilize both ATP and CTP to activate PPA in vitro. The results of isothermal titration calorimetry indicate that PPA, CTP, and ATP could bind to Cab2 individually, with PPA having the highest binding affinity. To provide further insight into the catalytic mechanism of Cab2, we determined the crystal structures of Cab2 and its complex with PPA, the reaction intermediate 4'-phosphopantothenoyl-CMP, the final reaction product PPC, and the product analogue phosphopantothenoylcystine. Except for PPA, all other ligands were generated in situ and present in the active-site pocket of Cab2. Structures of Cab2 in complex with ligands provide insight into substrates binding and its catalytic mechanism. Analysis of structures indicates that the carboxyl of PPA-moiety of ligands and the γ-amino group of Asn97 possess different conformations in these complex structures. The cysteine/cystine/serine selectivity assays for Cab2 indicate that the amino group rather than the thiol group of l-cysteine attacks the carbonyl of 4'-phosphopantothenoyl-CMP to form PPC. Based on structural and biochemical data, the catalytic mechanism of Cab2 was proposed for the first time.

Fabrication of p-Type ZnO:N Films by Oxidizing Zn3N2 Films in Oxygen Plasma at Low Temperature.

The oxygen vacancy (VO) is known as the main compensation center in p-type ZnO, which leads to the difficulty of fabricating high-quality p-type ZnO. To reduce the oxygen vacancies, we oxidized Zn3N2 films in oxygen plasma and successfully prepared p-type ZnO:N films at temperatures ranging from room temperature to 300 °C. The films were characterized by X-ray diffraction (XRD), non-Rutherford backscattering (non-RBS) spectroscopy, X-ray photoelectron spectroscopy, photoluminescence spectrum, and Hall Effect. The results show that the nitrogen atoms successfully substitute the oxygen sites in the ZnO:N films. The film prepared at room temperature exhibits the highest hole concentration of 6.22 × 1018 cm-3, and the lowest resistivity of 39.47 Ω∙cm. In all ZnO:N films, the VO defects are reduced significantly. At 200 °C, the film holds the lowest value of VO defects and the strongest UV emission. These results imply that oxygen plasma is very efficient in reducing VO defects in p-type ZnO:N films, and could greatly reduce the reaction temperature. This method is significant for the development of ZnO-based optoelectronic devices.