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<p>Tpt1/TRPT1/KptA family proteins are evolutionarily conserved in all three domains of life. In fungi and plants, Tpt1 transfers 2’-PO<sub>4</sub><sup>2-</sup> from tRNA splice junction to NAD<sup>+</sup>, which is the final step of tRNA maturation and is critical for the function of tRNA. In mammals and bacteria, Tpt1-catalyzed reaction leads to 5’-end ADP ribosylation, a reversible chemical modification of nucleic acids. Based on in vivo and in vitro biochemical studies, a two-step catalytic mechanism has been established for Tpt1-catalyzed RNA 2’-PO<sub>4</sub><sup>2-</sup> transfer, including (i) the 2’-PO<sub>4</sub><sup>2-</sup> attacks NAD<sup>+</sup>, releasing nicotinamide and forming a 2’-phospho-ADP-ribosylated RNA (2’-p-ADPR-RNA) intermediate; and (ii) transesterification of the ADP-ribose 2”-OH to RNA 2’-PO<sub>4</sub><sup>2-</sup>, displacing the 2’-OH RNA and producing ADP-ribose-1”,2”-cyclic phosphate (Appr&gt;P). However, neither 2’-p-ADPR-RNA intermediate nor Appr&gt;P product has been captured in any reported Tpt1 structures. Here, we report a series of crystal structures of <i>T. kodakarensis</i> Tpt1 (<i>Tko</i>Tpt1), capturing the key 2’-p-ADPR-RNA intermediate. In addition, our structures also capture the 5’-p-ADPR-DNA intermediate and Appr&gt;P product. Structural analysis and in vitro catalytic assays revealed that <i>Tko</i>Tpt1 utilizes similar mechanism in 2’-PO<sub>4</sub><sup>2-</sup> and 5’-PO<sub>4</sub><sup>2-</sup> transfer. In conclusion, our structures reaffirm the catalytic mechanism of Tpt1-catalyzed phosphate transfer.</p>

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Crystal structures and snapshots along Tpt1-catalyzed phosphate transfer from nucleic acid to NAD+

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年12月4日

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  • Chulei Cao,
  • Jie Yang,
  • Weizhen Zhang,
  • Jiaqin Chen,
  • Yanqing Gao,
  • Yuqi Yao,
  • Yixi Zhang,
  • Huili Li,
  • Linxi Li,
  • Zhaorong Luo,
  • Chenxi Wang,
  • Guangli Tang,
  • Ruixue Cui,
  • Hehua Liu,
  • Qiyao Wang,
  • Zhen Huang,
  • Jinbiao Ma,
  • Jianhua Gan

摘要

Tpt1/TRPT1/KptA family proteins are evolutionarily conserved in all three domains of life. In fungi and plants, Tpt1 transfers 2’-PO42- from tRNA splice junction to NAD+, which is the final step of tRNA maturation and is critical for the function of tRNA. In mammals and bacteria, Tpt1-catalyzed reaction leads to 5’-end ADP ribosylation, a reversible chemical modification of nucleic acids. Based on in vivo and in vitro biochemical studies, a two-step catalytic mechanism has been established for Tpt1-catalyzed RNA 2’-PO42- transfer, including (i) the 2’-PO42- attacks NAD+, releasing nicotinamide and forming a 2’-phospho-ADP-ribosylated RNA (2’-p-ADPR-RNA) intermediate; and (ii) transesterification of the ADP-ribose 2”-OH to RNA 2’-PO42-, displacing the 2’-OH RNA and producing ADP-ribose-1”,2”-cyclic phosphate (Appr>P). However, neither 2’-p-ADPR-RNA intermediate nor Appr>P product has been captured in any reported Tpt1 structures. Here, we report a series of crystal structures of T. kodakarensis Tpt1 (TkoTpt1), capturing the key 2’-p-ADPR-RNA intermediate. In addition, our structures also capture the 5’-p-ADPR-DNA intermediate and Appr>P product. Structural analysis and in vitro catalytic assays revealed that TkoTpt1 utilizes similar mechanism in 2’-PO42- and 5’-PO42- transfer. In conclusion, our structures reaffirm the catalytic mechanism of Tpt1-catalyzed phosphate transfer.