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'-PO4DNA 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+)2structure highlights a two-metal mechanism, whereby: a ligase-bound "catalytic" Mg2+(H2O)5coordination complex lowers the pKaof 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)5complex lowers the lysine pKaand 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.
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065.
authors.forename: Mihaela-Carmen
authors.initials: MC
authors.lastname: Unciuleac
authors.affiliation: Structural Biology Program, Sloan-Kettering Institute, New York, NY 10065.
authors.forename: Yehuda
authors.initials: Y
authors.lastname: Goldgur
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065; s-shuman@ski.mskcc.org.
authors.forename: Stewart
authors.initials: S
authors.lastname: Shuman
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D011088:DNA Ligases / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D008670:Metals / Q000737:chemistry; D046912:Multiprotein Complexes / Q000737:chemistry*; D009243:NAD / Q000737:chemistry; D011487:Protein Conformation; D011130:RNA Ligase (ATP) / Q000737:chemistry* / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D008670:Metals; D046912:Multiprotein Complexes; D014764:Viral Proteins; D009243:NAD; D000255:Adenosine Triphosphate; D011088:DNA Ligases; C497118:LigA protein, E coli; D011130:RNA Ligase (ATP); C477285:bacteriophage T4 RNA ligase 1; D008239:Lysine
keywords: covalent nucleotidyltransferase; lysyl-AMP; metal catalysis