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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-N?)-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 ? phosphate; a second octahedral Mg2+ coordination complex bridges the ? and ? phosphates; and protein elements unique to Rnl1 engage the ? 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+ ? phosphate, but the ? 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.

SUBMITTER: Unciuleac MC 

PROVIDER: S-EPMC5347617 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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

Unciuleac Mihaela-Carmen MC   Goldgur Yehuda Y   Shuman Stewart S  

Proceedings of the National Academy of Sciences of the United States of America 20170221 10


Polynucleotide ligases comprise a ubiquitous superfamily of nucleic acid repair enzymes that join 3'-OH and 5'-PO<sub>4</sub> DNA or RNA ends. Ligases react with ATP or NAD<sup>+</sup> 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<sup>+</sup>-dependent DNA ligase family (<i>Escherichia coli</i> LigA), captured as thei  ...[more]

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