Structure Guided Understanding of NAD⁺ Recognition in Bacterial DNA Ligases

NAD⁺-dependent DNA ligases (LigA) are essential bacterial enzymes that catalyze phosphodiester bond formation during DNA replication and repair processes. Phosphodiester bond formation proceeds through a 3-step reaction mechanism. In the first step, the LigA adenylation domain interacts with NAD⁺ to form a covalent enzyme-AMP complex. Although it is well established that the specificity for binding of NAD⁺ resides within the adenylation domain, the precise recognition elements for the initial binding event remain unclear. We report here the structure of the adenylation domain from <i>Haemophilus influenzae</i> LigA. This structure is a first snapshot of a LigA-AMP intermediate with NAD⁺ bound to domain 1a in its open conformation. The binding affinities of NAD⁺ for adenylated and nonadenylated forms of the <i>H. influenzae</i> LigA adenylation domain were similar. The combined crystallographic and NAD⁺-binding data suggest that the initial recognition of NAD⁺ is via the NMN binding region in domain 1a of LigA

Antibacterial FabH Inhibitors with Mode of Action Validated in Haemophilus influenzae by in Vitro Resistance Mutation Mapping

Fatty acid biosynthesis is essential to bacterial growth in Gram-negative pathogens. Several small molecules identified through a combination of high-throughput and fragment screening were cocrystallized with FabH (β-ketoacyl-acyl carrier protein synthase III) from Escherichia coli and Streptococcus pneumoniae. Structure-based drug design was used to merge several scaffolds to provide a new class of inhibitors. After optimization for Gram-negative enzyme inhibitory potency, several compounds demonstrated antimicrobial activity against an efflux-negative strain of Haemophilus influenzae. Mutants resistant to these compounds had mutations in the FabH gene near the catalytic triad, validating FabH as a target for antimicrobial drug discovery

Selective Inhibitors of Bacterial t‑RNA-(N¹G37) Methyltransferase (TrmD) That Demonstrate Novel Ordering of the Lid Domain

The tRNA-(N¹G37) methyltransferase (TrmD) is essential for growth and highly conserved in both Gram-positive and Gram-negative bacterial pathogens. Additionally, TrmD is very distinct from its human orthologue TRM5 and thus is a suitable target for the design of novel antibacterials. Screening of a collection of compound fragments using Haemophilus influenzae TrmD identified inhibitory, fused thieno-pyrimidones that were competitive with <i>S</i>-adenosylmethionine (SAM), the physiological methyl donor substrate. Guided by X-ray cocrystal structures, fragment <b>1</b> was elaborated into a nanomolar inhibitor of a broad range of Gram-negative TrmD isozymes. These compounds demonstrated no activity against representative human SAM utilizing enzymes, PRMT1 and SET7/9. This is the first report of selective, nanomolar inhibitors of TrmD with demonstrated ability to order the TrmD lid in the absence of tRNA