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  • 标题:Two-metal versus one-metal mechanisms of lysine adenylylation by ATP-dependent and NAD+-dependent polynucleotide ligases
  • 本地全文:下载
  • 作者:Mihaela-Carmen Unciuleac ; Yehuda Goldgur ; Stewart Shuman
  • 期刊名称:Proceedings of the National Academy of Sciences
  • 印刷版ISSN:0027-8424
  • 电子版ISSN:1091-6490
  • 出版年度:2017
  • 卷号:114
  • 期号:10
  • 页码:2592-2597
  • DOI:10.1073/pnas.1619220114
  • 语种:English
  • 出版社:The National Academy of Sciences of the United States of America
  • 摘要:Polynucleotide ligases comprise a ubiquitous superfamily of nucleic acid repair enzymes that join 3′-OH and 5′-PO4 DNA or RNA ends. Ligases react with ATP or NAD+ and a divalent cation cofactor to form a covalent enzyme-(lysine-Nζ)–adenylate intermediate. Here, we report crystal structures of the founding members of the ATP-dependent RNA ligase family (T4 RNA ligase 1; Rnl1) and the NAD+-dependent DNA ligase family ( Escherichia coli LigA), captured as their respective Michaelis complexes, which illuminate distinctive catalytic mechanisms of the lysine adenylylation reaction. The 2.2-Å Rnl1•ATP•(Mg2+)2 structure highlights a two-metal mechanism, whereby: a ligase-bound “catalytic” Mg2+(H2O)5 coordination complex lowers the p K a of the lysine nucleophile and stabilizes the transition state of the ATP α phosphate; a second octahedral Mg2+ coordination complex bridges the β and γ phosphates; and protein elements unique to Rnl1 engage the γ phosphate and associated metal complex and orient the pyrophosphate leaving group for in-line catalysis. By contrast, the 1.55-Å LigA•NAD+•Mg2+ structure reveals a one-metal mechanism in which a ligase-bound Mg2+(H2O)5 complex lowers the lysine p K a and engages the NAD+ α phosphate, but the β phosphate and the nicotinamide nucleoside of the nicotinamide mononucleotide (NMN) leaving group are oriented solely via atomic interactions with protein elements that are unique to the LigA clade. The two-metal versus one-metal dichotomy demarcates a branchpoint in ligase evolution and favors LigA as an antibacterial drug target.
  • 关键词:metal catalysis ; covalent nucleotidyltransferase ; lysyl-AMP
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