Structure-guided Mutational Analysis of the Nucleotidyltransferase Domain of Escherichia coli DNA Ligase (LigA).

The Journal of Biological Chemistry
Li Kai WangS Shuman

Abstract

NAD(+)-dependent DNA ligases (LigA) are ubiquitous in bacteria, where they are essential for growth and present attractive targets for antimicrobial drug discovery. LigA has a distinctive modular structure in which a nucleotidyltransferase catalytic domain is flanked by an upstream NMN-binding module and by downstream OB-fold, zinc finger, helix-hairpin-helix, and BRCT domains. Here we conducted a structure-function analysis of the nucleotidyltransferase domain of Escherichia coli LigA, guided by the crystal structure of the LigA-DNA-adenylate intermediate. We tested the effects of 29 alanine and conservative mutations at 15 amino acids on ligase activity in vitro and in vivo. We thereby identified essential functional groups that coordinate the reactive phosphates (Arg(136)), contact the AMP adenine (Lys(290)), engage the phosphodiester backbone flanking the nick (Arg(218), Arg(308), Arg(97) plus Arg(101)), or stabilize the active domain fold (Arg(171)). Finer analysis of the mutational effects revealed step-specific functions for Arg(136), which is essential for the reaction of LigA with NAD(+) to form the covalent ligase-AMP intermediate (step 1) and for the transfer of AMP to the nick 5'-PO(4) to form the DNA-adenylate inte...Continue Reading

References

Nov 1, 1967·Proceedings of the National Academy of Sciences of the United States of America·J W LittleM Gellert
Sep 1, 1968·Proceedings of the National Academy of Sciences of the United States of America·B M OliveraI R Lehman
Oct 1, 1971·Proceedings of the National Academy of Sciences of the United States of America·R I Gumport, I R Lehman
Jan 8, 1999·Journal of Molecular Biology·D J Timson, D B Wigley
Jun 16, 1999·Structure·M R SingletonD B Wigley
Jul 19, 2001·The Journal of Biological Chemistry·V SriskandaS Shuman
Jan 10, 2002·The Journal of Biological Chemistry·Verl Sriskanda, Stewart Shuman
Mar 4, 2003·The Journal of Biological Chemistry·Shenmin YinStewart Shuman
Jul 23, 2004·FEMS Microbiology Letters·Hyo Jeong JeonSuk-Tae Kwon
Dec 8, 2004·Current Opinion in Structural Biology·Stewart Shuman, Christopher D Lima
Nov 3, 2005·The Journal of Biological Chemistry·Kamel El OmariDavid K Stammers
Jul 18, 2006·Virology·Delphine Benarroch, Stewart Shuman
Oct 28, 2006·RNA·Li Kai WangStewart Shuman
May 1, 2007·Molecular Cell·Jayakrishnan NandakumarStewart Shuman
Jul 10, 2007·Nature Structural & Molecular Biology·Pravin A NairStewart Shuman
Jun 3, 2008·The Journal of Biological Chemistry·Li Kai WangStewart Shuman

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Citations

May 16, 2012·Antimicrobial Agents and Chemotherapy·Steven D PodosMichael J Pucci
Feb 10, 2016·Nucleic Acids Research·Mathieu Chauleau, Stewart Shuman
Feb 23, 2017·Proceedings of the National Academy of Sciences of the United States of America·Mihaela-Carmen UnciuleacStewart Shuman
Sep 2, 2016·Bioscience Reports·Giulia PergolizziRichard Peter Bowater
May 1, 2021·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Arqam AlomariDarren M Gowers
Jan 11, 2012·ACS Chemical Biology·Sushmita D LahiriScott D Mills

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