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Title: 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 (Escherichia coliLigA), 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 aremore » unique to the LigA clade. We conclude, the two-metal versus one-metal dichotomy demarcates a branchpoint in ligase evolution and favors LigA as an antibacterial drug target.« less

Authors:
 [1];  [1];  [1]
+ Show Author Affiliations
  1. Sloan–Kettering Inst., New York, NY (United States)
Publication Date:
Research Org.:
Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
National Institutes of Health (NIH); National Cancer Institute (NCI); National Institute of General Medical Sciences; USDOE
OSTI Identifier:
1430349
Grant/Contract Number:  
GM63611; P30-CA008748; P41GM103403; P41GM103473; P41GM111244; HEI S10RR029205; AC02-06CH11357; AC02-98CH10886; SC0012704
Resource Type:
Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 114; Journal Issue: 10; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; metal catalysis; covalent nucleotidyltransferase; lysyl-AMP

Citation Formats

Unciuleac, Mihaela-Carmen, Goldgur, Yehuda, and Shuman, Stewart. Two-metal versus one-metal mechanisms of lysine adenylylation by ATP-dependent and NAD+-dependent polynucleotide ligases. United States: N. p., 2017. Web. doi:10.1073/pnas.1619220114.
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Unciuleac, Mihaela-Carmen, Goldgur, Yehuda, & Shuman, Stewart. Two-metal versus one-metal mechanisms of lysine adenylylation by ATP-dependent and NAD+-dependent polynucleotide ligases. United States. https://doi.org/10.1073/pnas.1619220114
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Unciuleac, Mihaela-Carmen, Goldgur, Yehuda, and Shuman, Stewart. Tue . "Two-metal versus one-metal mechanisms of lysine adenylylation by ATP-dependent and NAD+-dependent polynucleotide ligases". United States. https://doi.org/10.1073/pnas.1619220114. https://www.osti.gov/servlets/purl/1430349.
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@article{osti_1430349,
title = {Two-metal versus one-metal mechanisms of lysine adenylylation by ATP-dependent and NAD+-dependent polynucleotide ligases},
author = {Unciuleac, Mihaela-Carmen and Goldgur, Yehuda and Shuman, Stewart},
abstractNote = {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 coliLigA), 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. We conclude, the two-metal versus one-metal dichotomy demarcates a branchpoint in ligase evolution and favors LigA as an antibacterial drug target.},
doi = {10.1073/pnas.1619220114},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 10,
volume = 114,
place = {United States},
year = {Tue Feb 21 00:00:00 EST 2017},
month = {Tue Feb 21 00:00:00 EST 2017}
}
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https://doi.org/10.1073/pnas.1619220114
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