title: The kinetic mechanism of S. pneumoniae DNA ligase and inhibition by adenosine-based antibacterial compounds.
abstract: The NAD-dependent DNA ligase is an excellent target for the discovery of antibacterial agents with a novel mode of action. In this work the DNA ligase from Streptococcus pneumoniae was investigated for its steady-state kinetic parameters and inhibition by compounds with an adenosine substructure. Inhibition by substrate DNA that was observed in the enzyme turnover experiments was verified by direct binding measurements using isothermal titration calorimetry (ITC). The substrate-inhibited enzyme form was identified as deadenylated DNA ligase. The binding potencies of 2-(butylsulfanyl) adenosine and 2-(cyclopentyloxy) adenosine were not significantly affected by the presence of the enzyme-bound DNA substrate. Finally, a mutant protein was prepared that was known to confer resistance to the adenosine compounds' antibacterial activity. The mutant protein was shown to have little catalytic impairment yet it was less susceptible to adenosine compound inhibition.
authors: Infection Innovative Medicines Unit, AstraZeneca R&D Boston, Waltham, MA 02451, USA.
authors: Haris
authors: H
authors: Jahić
authors: Ce Feng
authors: CF
authors: Liu
authors: Jason
authors: J
authors: Thresher
authors: Stephania
authors: S
authors: Livchak
authors: Hongming
authors: H
authors: Wang
authors: David E
authors: DE
authors: Ehmann
mesh_terms: D000241:Adenosine / Q000737:chemistry / Q000494:pharmacology*; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D001483:Base Sequence; D002151:Calorimetry; D011088:DNA Ligases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D055808:Drug Discovery; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D013296:Streptococcus pneumoniae / Q000201:enzymology*
chemical_list: D000900:Anti-Bacterial Agents; D017931:DNA Primers; D004791:Enzyme Inhibitors; D011088:DNA Ligases; D000241:Adenosine