The PhoU protein in bacteria plays a role in maintaining phosphate homeostasis by regulating the Pho regulon. Recent studies showed that PhoU is essential for normal growth and is also involved in persister formation. PhoU is a potential target for overcoming drug tolerance of pathogenic bacteria. However, the exact mechanism of PhoU functions is still unknown. Here we have determined the crystal structure of PhoU from Pseudomonas aeruginosa at 2.28 Å resolution by Se SAD method. P. aeruginosa PhoU exists as a dimer in the crystals. A monomer of P. aeruginosa PhoU consists of six alpha-helices, which form two similar helical bundles. P. aeruginosa PhoU shares four conserved sequence motifs. Interestingly, P. aeruginosa PhoU has distinct features in some loops and the surface charge distribution. Two monomers of P. aeruginosa PhoU dimerize in a slightly different manner to those of other PhoU proteins. The present structure of PhoU from a bacterial pathogen may be useful for the antibacterial drug discovery.

Helicobacter pylori infection is the main cause of chronic gastritis, gastric mucosal atrophy, peptic ulcer, and some forms of gastric cancer. There has been considerable interest in strain-specific genes found outside of the cag pathogenicity island, especially genes in the plasticity regions of H. pylori. In H. pylori strain J99, the plasticity region contains 48 genes ranging from jhp0914 to jhp0961. Because little is known about many of these genes in the plasticity region, further studies are necessary to elucidate their roles in H. pylori-associated pathogenesis. The JHP933 protein, encoded by the jhp0933 gene in the plasticity region of H. pylori J99, is one of the prevalently expressed proteins in some gastritis and peptic ulcer patients. However, its structure and function remain unknown. Here, we have determined the crystal structure of JHP933, revealing the first two-domain architecture of DUF1814 family. The N-terminal domain has the nucleotidyltransferase fold and the C-terminal domain is a helix bundle. Structural similarity of JHP933 to known nucleotidyltransferases is very remote, suggesting that it may function as a novel nucleotidyltransferase. It is expected that this study will facilitate functional characterization of JHP933 to obtain an insight into its role in pathogenesis by the H. pylori plasticity region.

The toxin-antitoxin (TA) systems widely spread among bacteria and archaea are important for antibiotic resistance and virulence. The bacterial kingdom uses TA systems to adjust the global level of gene expression and translation through RNA degradation. The HP0892-HP0893 and HP0894-HP0895 toxin-antitoxin systems are the only two known TA systems belonging to Helicobacter pylori. In both of these TA systems, the antitoxin binds and inhibits the toxin and regulates the transcription of the TA operon. However, the precise molecular basis for interaction with substrate or antitoxin and the mechanism of mRNA cleavage remains unclear. Therefore, here an attempt was made to shed some light on the mechanism behind the TA system of HP0892-HP0893 and HP0894-HP0895. Here, we present the crystal structures of apo- and copper-bound HP0894 at 1.28 Å and 1.89 Å, respectively, and the crystal structure of the zinc-bound HP0892 toxin at 1.8 Å resolution. Reorientation of residues involving the mRNase active site was shown. Through the combined approach of structural analysis along with isothermal calorimetry studies and structural homology search, the amino acids involved in mRNase active site were monitored. In the mRNase active site of HP0894 toxin, His84 acts as a catalytic residue and reorients itself acting as a general acid in an acid-base catalysis reaction, while His47 and His60 stabilize the transition state. Glu58 acts as a general base, and substrate reorientation is caused by Phe88. In the mRNase active site of HP0892 toxin, the most catalytically important residue, His86, reorients itself to exhibit RNase activity while Glu58 acts as a general base. His47 and His60 are considered to be involved in enzymatic activity. Glu58 and Asp64 are involved in substrate binding and specific sequence recognition. The mutational constructs were used for isothermal calorimetric studies to analyze the effect of catalytic residues.