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

Abstract

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 (<i>Escherichia coli</i> 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<i>K</i>_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<i>K</i>_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.