5TT5

Two-metal versus one-metal mechanisms of lysine adenylylation by ATP-dependent and NAD(+)-dependent polynucleotide ligases.

(2017) Proc Natl Acad Sci U S A 114: 2592-2597

• PubMed: 28223499 Search on PubMedSearch on PubMed Central
• DOI: https://doi.org/10.1073/pnas.1619220114
• Primary Citation of Related Structures:  
  5TT5, 5TT6


• PubMed Abstract: 
  

  Polynucleotide ligases comprise a ubiquitous superfamily of nucleic acid repair enzymes that join 3'-OH and 5'-PO ₄ 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•(Mg ²⁺ ) ₂ structure highlights a two-metal mechanism, whereby: a ligase-bound "catalytic" Mg ²⁺ (H ₂ O) 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 Mg ²⁺ 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 ⁺ •Mg ²⁺ structure reveals a one-metal mechanism in which a ligase-bound Mg ²⁺ (H ₂ O) 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.

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Organizational Affiliation: 

Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065.