Conserved residues in domain Ia are required for the reaction of Escherichia coli DNA ligase with NAD+.
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'-PO(4) nicks and the reaction with NAD(+) to form ligase-adenylate 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'-PO(4) 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.
Sriskanda
Verl
V
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Shuman
Stewart
S
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D011088:DNA Ligases / Q000737:chemistry / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology*; D017353:Gene Deletion; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D009243:NAD / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry
D008024:Ligands; D009243:NAD; D001224:Aspartic Acid; D014443:Tyrosine; D006639:Histidine; D011088:DNA Ligases; D000409:Alanine