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TatD has been thoroughly investigated as a DNA-repair enzyme and an apoptotic nuclease, and still-unknown TatD-related DNases are considered to play crucial cellular roles. However, studies of TatD from Gram-positive bacteria have been hindered by an absence of atomic detail and the resulting inability to determine function from structure. In this study, an X-ray crystal structure of SAV0491, which is the TatD enzyme from the Gram-positive bacterium Staphylococcus aureus (SaTatD), is reported at a high resolution of 1.85 Å with a detailed atomic description. Although SaTatD has the common TIM-barrel fold shared by most TatD-related homologs, and PDB entry 2gzx shares 100% sequence identity with SAV0491, the crystal structure of SaTatD revealed a unique binding mode of two phosphates interacting with two Ni2+ ions. Through a functional study, it was verified that SaTatD has Mg2+-dependent nuclease activity as a DNase and an RNase. In addition, structural comparison with TatD homologs and the identification of key residues contributing to the binding mode of Ni2+ ions and phosphates allowed mutational studies to be performed that revealed the catalytic mechanism of SaTatD. Among the key residues composing the active site, the acidic residues Glu92 and Glu202 had a critical impact on catalysis by SaTatD. Furthermore, based on the binding mode of the two phosphates and structural insights, a putative DNA-binding mode of SaTatD was proposed using in silico docking. Overall, these findings may serve as a good basis for understanding the relationship between the structure and function of TatD proteins from Gram-positive bacteria and may provide critical insights into the DNA-binding mode of SaTatD.

HP0268 is a conserved, uncharacterized protein from Helicobacter pylori. Here, we determined the solution structure of HP0268 using three-dimensional nuclear magnetic resonance (NMR) spectroscopy, revealing that this protein is structurally most similar to a small MutS-related (SMR) domain that exhibits nicking endonuclease activity. We also demonstrated for the first time that HP0268 is a nicking endonuclease and a purine-specific ribonuclease through gel electrophoresis and fluorescence spectroscopy. The nuclease activities for DNA and RNA were maximally increased by Mn2+ and Mg2+ ions, respectively, and decreased by Cu2+ ions. Using NMR chemical shift perturbations, the metal and nucleotide binding sites of HP0268 were determined to be spatially divided but close to each other. The lysine residues (Lys7, Lys11 and Lys43) are clustered and form the nucleotide binding site. Moreover, site-directed mutagenesis was used to define the catalytic active site of HP0268, revealing that this site contains two acidic residues, Asp50 and Glu54, in the metal binding site. The nucleotide binding and active sites are not conserved in the structural homologues of HP0268. This study will contribute to improving our understanding of the structure and functionality of a wide spectrum of nucleases.

Oxidative stresses, such as reactive oxygen species, reactive electrophilic species, reactive nitrogen species, and reactive chlorine species, can damage cellular components, leading to cellular malfunction and death. In response to oxidative stress, bacteria have evolved redox-responsive sensors that enable them to simultaneously monitor and eradicate potential oxidative stress. Specifically, redox-sensing transcription regulators react to oxidative stress by means of modifying the thiol groups of cysteine residues, functioning as part of an efficient survival mechanism for many bacteria. In general, oxidative molecules can induce changes in the three-dimensional structures of redox sensors, which, in turn, affects the transcription of specific genes in detoxification pathways and defense mechanisms. Moreover, pathogenic bacteria utilize these redox sensors for adaptation and to evade subsequent oxidative attacks from host immune defense. For this reason, the redox sensors of pathogenic bacteria are potential antibiotic targets. Understanding the regulatory mechanisms of thiol-based redox sensors in bacteria will provide insight and knowledge into the discovery of new antibiotics.

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