{"title":"恶性疟原虫核苷酸管家蛋白 HAM1 的结构功能分析。","authors":"Debanjan Saha, Atanu Pramanik, Aline Freville, Asim Azhar Siddiqui, Uttam Pal, Chinmoy Banerjee, Shiladitya Nag, Subhashis Debsharma, Saikat Pramanik, Somnath Mazumder, Nakul C. Maiti, Saumen Datta, Christiaan van Ooij, Uday Bandyopadhyay","doi":"10.1111/febs.17216","DOIUrl":null,"url":null,"abstract":"<p>Non-canonical nucleotides, generated as oxidative metabolic by-products, significantly threaten the genome integrity of <i>Plasmodium falciparum</i> and thereby, their survival, owing to their mutagenic effects. <i>Pf</i>HAM1, 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 <i>Pf</i>HAM1, 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. <i>Pf</i>HAM1 exhibited Mg<sup>++</sup>-dependent pyrophosphohydrolase activity and the highest binding affinity to dITP compared to other non-canonical nucleotides as measured by isothermal titration calorimetry. Modifying the <i>pfham1</i> genomic locus followed by live-cell imaging of expressed mNeonGreen-tagged <i>Pf</i>HAM1 demonstrated its ubiquitous presence in the cytoplasm across erythrocytic stages with greater expression in trophozoites and schizonts. Interestingly, CRISPR-Cas9/DiCre recombinase-guided <i>pfham1</i>-null <i>P. falciparum</i> 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 <i>Pf</i>HAM1, an atypical nucleotide-cleansing enzyme in <i>P. falciparum.</i></p>","PeriodicalId":94226,"journal":{"name":"The FEBS journal","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2024-07-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Structure–function analysis of nucleotide housekeeping protein HAM1 from human malaria parasite Plasmodium falciparum\",\"authors\":\"Debanjan Saha, Atanu Pramanik, Aline Freville, Asim Azhar Siddiqui, Uttam Pal, Chinmoy Banerjee, Shiladitya Nag, Subhashis Debsharma, Saikat Pramanik, Somnath Mazumder, Nakul C. Maiti, Saumen Datta, Christiaan van Ooij, Uday Bandyopadhyay\",\"doi\":\"10.1111/febs.17216\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>Non-canonical nucleotides, generated as oxidative metabolic by-products, significantly threaten the genome integrity of <i>Plasmodium falciparum</i> and thereby, their survival, owing to their mutagenic effects. <i>Pf</i>HAM1, 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 <i>Pf</i>HAM1, 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. <i>Pf</i>HAM1 exhibited Mg<sup>++</sup>-dependent pyrophosphohydrolase activity and the highest binding affinity to dITP compared to other non-canonical nucleotides as measured by isothermal titration calorimetry. Modifying the <i>pfham1</i> genomic locus followed by live-cell imaging of expressed mNeonGreen-tagged <i>Pf</i>HAM1 demonstrated its ubiquitous presence in the cytoplasm across erythrocytic stages with greater expression in trophozoites and schizonts. Interestingly, CRISPR-Cas9/DiCre recombinase-guided <i>pfham1</i>-null <i>P. falciparum</i> 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 <i>Pf</i>HAM1, an atypical nucleotide-cleansing enzyme in <i>P. falciparum.</i></p>\",\"PeriodicalId\":94226,\"journal\":{\"name\":\"The FEBS journal\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2024-07-14\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"The FEBS journal\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://onlinelibrary.wiley.com/doi/10.1111/febs.17216\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"The FEBS journal","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1111/febs.17216","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
Structure–function analysis of nucleotide housekeeping protein HAM1 from human malaria parasite Plasmodium falciparum
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.