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Non‐canonical nucleotides, generated as oxidative metabolic by‐products, significantly threaten the genome integrity of Plasmodium falciparum and thereby, their survival, owing to their mutagenic effects. PfHAM1, an evolutionarily conserved inosine/xanthosine triphosphate pyrophosphohydrolase, maintains nucleotide homeostasis in the malaria parasite by removing non‐canonical nucleotides, although structure–function intricacies are hitherto poorly reported. Here, we report the X‐ray crystal structure of PfHAM1, which revealed a homodimeric structure, additionally validated by size‐exclusion chromatography–multi‐angle light scattering analysis. The two monomeric units in the dimer were aligned in a parallel fashion, and critical residues associated with substrate and metal binding were identified, wherein a notable structural difference was observed in the β‐sheet main frame compared to human inosine triphosphate pyrophosphatase. PfHAM1 exhibited Mg++‐dependent pyrophosphohydrolase activity and the highest binding affinity to dITP compared to other non‐canonical nucleotides as measured by isothermal titration calorimetry. Modifying the pfham1 genomic locus followed by live‐cell imaging of expressed mNeonGreen‐tagged PfHAM1 demonstrated its ubiquitous presence in the cytoplasm across erythrocytic stages with greater expression in trophozoites and schizonts. Interestingly, CRISPR‐Cas9/DiCre recombinase‐guided pfham1‐null P. falciparum survived in culture under standard growth conditions, indicating its assistive role in non‐canonical nucleotide clearance during intra‐erythrocytic stages. This is the first comprehensive structural and functional report of PfHAM1, an atypical nucleotide‐cleansing enzyme in P. falciparum. [ABSTRACT FROM AUTHOR]

ExoT belongs to the family of type 3 secretion system (T3 SS) effector toxins in Pseudomonas aeruginosa, known to be one of the major virulence determinant toxins that cause chronic and acute infections in immuno-compromised individuals, burn victims and cystic fibrosis patients. Here, we report the X-ray crystal structure of the amino terminal fragment of effector toxin ExoT, in complex with full-length homodimeric chaperone SpcS at 2.1 Å resolution. The full-length dimeric chaperone SpcS has the conserved α-β-β-β-α-β-β-α fold of class I chaperones, the characteristic hydrophobic patches for binding effector proteins and a conserved polar cavity at the dimeric interface. The stable crystallized amino terminal fragment of ExoT consists of a chaperone binding domain and a membrane localization domain that wraps around the dimeric chaperone. Site-directed mutagenesis experiments and a molecular dynamics study complement each other in revealing Asn65, Phe67 and Trp88 as critical dimeric interfacial residues that can strongly influence the effector-chaperone interactions. Database The atomic coordinates and structure factors of ExoT-SpcS complex (code ) have been deposited in the Protein Data Bank Japan (PDBj), Institute for Protein Research, Osaka University. [ABSTRACT FROM AUTHOR]