Structure-guided mutational analysis of the nucleotidyltransferase domain of Escherichia coli DNA Ligase (LigA) Journal Article
| Authors: | Wang, L. K.; Zhu, H.; Shuman, S. |
| Article Title: | Structure-guided mutational analysis of the nucleotidyltransferase domain of Escherichia coli DNA Ligase (LigA) |
| Abstract: | NAD<sup>+</sup>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<sup>136</sup>), contact the AMP adenine (Lys<sup>290</sup>), engage the phosphodiester backbone flanking the nick (Arg<sup>218</sup>, Arg<sup>308</sup>, Arg<sup>97</sup> plus Arg<sup>101</sup>), or stabilize the active domain fold (Arg171). Finer analysis of the mutational effects revealed step-specific functions for Arg<sup>136</sup>, which is essential for the reaction of LigA with NAD<sup>+</sup> to form the covalent ligase-AMP intermediate (step 1) and for the transfer of AMP to the nick 5′-PO<sub>4</sub> to form the DNA-adenylate intermediate (step 2) but is dispensable for phosphodiester formation at a preadenylylated nick (step 3). © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. |
| Keywords: | controlled study; gene mutation; nonhuman; protein domain; protein function; genes; protein stability; in vivo study; in vitro study; enzyme activity; structure activity relation; mutational analysis; amines; dna; escherichia coli; nucleic acids; crystal structure; alanine; amino acids; organic acids; protein structure; zinc; polydeoxyribonucleotide synthase; in-vitro; enzymes; arginine; dna ligase; dna ligases; adenylate; functional groups; adenine; antimicrobial drug discovery; catalytic domains; ligase activity; modular structures; mutational effects; phosphodiester; reactive phosphates; structure-function analysis; zinc finger; nucleotidyltransferase; liga |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 284 |
| Issue: | 13 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2009-03-27 |
| Start Page: | 8486 |
| End Page: | 8494 |
| Language: | English |
| DOI: | 10.1074/jbc.M808476200 |
| PROVIDER: | scopus |
| PMCID: | PMC2659207 |
| PUBMED: | 19150981 |
| DOI/URL: | |
| Notes: | --- - "Export Date: 30 November 2010" - "CODEN: JBCHA" - "Source: Scopus" |
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