Structural and functional studies of SAV1707 from Staphylococcus aureus elucidate its distinct metal-dependent activity and a crucial residue for catalysis.

The metallo-ß-lactamase fold is the most abundant metal-binding domain found in two major kingdoms: bacteria and archaea. Despite the rapid growth in genomic information, most of these enzymes, which may play critical roles in cellular metabolism, remain uncharacterized in terms of structure and function. In this study, X-ray crystal structures of SAV1707, a hypothetical metalloenzyme from Staphylococcus aureus, and its complex with cAMP are reported at high resolutions of 2.05 and 1.55 Å, respectively, with a detailed atomic description. Through a functional study, it was verified that SAV1707 has Ni2+-dependent phosphodiesterase activity and Mn2+-dependent endonuclease activity, revealing a different metal selectivity depending on the reaction. In addition, the crystal structure of cAMP-bound SAV1707 shows a unique snapshot of cAMP that reveals the binding mode of the intermediate, and a key residue Phe511 that forms π-π interactions with cAMP was verified as contributing to substrate recognition by functional studies of its mutant. Overall, these findings characterized the relationship between the structure and function of SAV1707 and may provide further understanding of metalloenzymes possessing the metallo-ß-lactamase fold.

Crystal structures of human NSDHL and development of its novel inhibitor with the potential to suppress EGFR activity.

NAD(P)-dependent steroid dehydrogenase-like (NSDHL), an essential enzyme in human cholesterol synthesis and a regulator of epidermal growth factor receptor (EGFR) trafficking pathways, has attracted interest as a therapeutic target due to its crucial relevance to cholesterol-related diseases and carcinomas. However, the development of pharmacological agents for targeting NSDHL has been hindered by the absence of the atomic details of NSDHL. In this study, we reported two X-ray crystal structures of human NSDHL, which revealed a detailed description of the coenzyme-binding site and the unique conformational change upon the binding of a coenzyme. A structure-based virtual screening and biochemical evaluation were performed and identified a novel inhibitor for NSDHL harboring suppressive activity towards EGFR. In EGFR-driven human cancer cells, treatment with the potent NSDHL inhibitor enhanced the antitumor effect of an EGFR kinase inhibitor. Overall, these findings could serve as good platforms for the development of therapeutic agents against NSDHL-related diseases.

A structural study of TatD from Staphylococcus aureus elucidates a putative DNA-binding mode of a Mg2+-dependent nuclease.

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 Šwith 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.

Crystal structure of proteolyzed VapBC and DNA-bound VapBC from Salmonella enterica Typhimurium LT2 and VapC as a putative Ca2+ -dependent ribonuclease.

Bacterial toxin-antitoxin (TA) system has gained attention for its essential roles in cellular maintenance and survival under harsh environmental conditions such as nutrient deficiency and antibiotic treatment. There are at least 14 TA systems in Salmonella enterica serovar Typhimurium LT2, a pathogenic bacterium, and none of the structures of these TA systems have been determined. We determined the crystal structure of the VapBC TA complex from S. Typhimurium LT2 in proteolyzed and DNA-bound forms at 2.0 Å and 2.8 Å resolution, respectively. The VapC toxin possesses a pilT N-terminal domain (PIN-domain) that shows ribonuclease activity, and the VapB antitoxin has an AbrB-type DNA binding domain. In addition, the structure revealed details of interaction mode between VapBC and the cognate promoter DNA, including the inhibition of VapC by VapB and linear conformation of bound DNA in the VapBC complex. The complexation of VapBC with the linear DNA is not consistent with known structures of VapBC homologs in complex with bent DNA. We also identified VapC from S. Typhimurium LT2 as a putative Ca2+ -dependent ribonuclease, which differs from previous data showing that VapC homologs have Mg2+ or Mn2+ -dependent ribonuclease activities. The present studies could provide structural understanding of the physiology of VapBC systems and foundation for the development of new antibiotic drugs against Salmonella infection.