Structure Guided Understanding of NAD⁺ Recognition in Bacterial DNA Ligases

Abstract

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