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 pKa 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 pKa 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.
|Number of pages||6|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|State||Published - 7 Mar 2017|
- Covalent nucleotidyltransferase
- Metal catalysis