Conserved residues in domain Ia are required for the reaction of Escherichia coli DNA ligase with NAD+ Journal Article
| Authors: | Sriskanda, V.; Shuman, S. |
| Article Title: | Conserved residues in domain Ia are required for the reaction of Escherichia coli DNA ligase with NAD+ |
| Abstract: | NAD+-dependent DNA ligases are present in all bacteria and are essential for growth. Their unique substrate specificity compared with ATP-dependent human DNA ligases recommends the NAD+ ligases as targets for the development of new broad-spectrum antibiotics. A plausible strategy for drug discovery is to identify the structural components of bacterial DNA ligase that interact with NAD+ and then to isolate small molecules that recognize these components and thereby block the binding of NAD+ to the ligase. The limitation to this strategy is that the structural determinants of NAD+ specificity are not known. Here we show that reactivity of Escherichia coli DNA ligase (LigA) with NAD+ requires N-terminal domain Ia, which is unique to, and conserved among, NAD+ ligases but absent from ATP-dependent ligases. Deletion of domain Ia abolished the sealing of 3′-OH/5′-PO4 nicks and the reaction with NAD+ to form ligaseadenylate but had no effect on phosphodiester formation at a preadenylated nick. Alanine substitutions at conserved residues within domain Ia either reduced (His-23, Tyr-35) or abolished (Tyr-22, Asp-32, Asp-36) sealing of a 5′-PO4 nick and adenylyl transfer from NAD+ without affecting ligation of pre-formed DNA-adenylate. We suggest that these five side chains comprise a binding site for the nicotinamide mononucleotide moiety of NAD+. Structure-activity relationships were clarified by conservative substitutions. |
| Keywords: | antibiotic agent; gene deletion; mutation; nonhuman; protein conformation; protein domain; models, biological; amino acid substitution; protein protein interaction; protein binding; dose-response relationship, drug; structure-activity relationship; tyrosine; bacteria (microorganisms); time factors; dna; amino acid sequence; molecular sequence data; sequence homology, amino acid; amino terminal sequence; kinetics; escherichia coli; ligands; binding site; protein structure, tertiary; binding sites; conformational transition; alanine; adenosine triphosphate; polydeoxyribonucleotide synthase; enzyme specificity; biochemistry; aspartic acid; genetic conservation; nad; substrates; histidine; nicotinamide adenine dinucleotide; dna ligases; bacterial growth; adenosine phosphate; adenylation; antibiotics; negibacteria; priority journal; article; nicotinamide nucleotide |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 277 |
| Issue: | 12 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2002-03-22 |
| Start Page: | 9695 |
| End Page: | 9700 |
| Language: | English |
| DOI: | 10.1074/jbc.M111164200 |
| PUBMED: | 11781321 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Export Date: 14 November 2014 -- Source: Scopus |
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