DNA ligases are important enzymes required for cellular processes such as DNA replication, recombination, and repair. NAD ؉ -dependent DNA ligases are essentially restricted to eubacteria, thus constituting an attractive target in the development of novel antibiotics. Although such a project might involve the systematic testing of a vast number of chemical compounds, it can essentially gain from the preliminary deciphering of the conformational stability and structural perturbations associated with the formation of the catalytically active adenylated enzyme. We have, therefore, investigated the adenylation-induced conformational changes in the mesophilic Escherichia coli and thermophilic Thermus scotoductus NAD ؉ -DNA ligases, and the resistance of these enzymes to thermal and chemical (guanidine hydrochloride) denaturation. Our results clearly demonstrate that anchoring of the cofactor induces a conformational rearrangement within the active site of both mesophilic and thermophilic enzymes accompanied by their partial compaction. Furthermore, the adenylation of enzymes increases their resistance to thermal and chemical denaturation, establishing a thermodynamic link between cofactor binding and conformational stability enhancement. Finally, guanidine hydrochloride-induced unfolding of NAD ؉ -dependent DNA ligases is shown to be a complex process that involves accumulation of at least two equilibrium intermediates, the molten globule and its precursor.DNA ligases form a large family of evolutionarily related proteins that play important roles in a wide range of DNA transactions, including chromosomal DNA replication, DNA repair, and DNA recombination in all three kingdoms of life (1). Cofactor requirements divide the ligases into two subfamilies, the NAD ϩ -dependent DNA ligases and the ATP-dependent DNA ligases. Regardless of their energy source, they catalyze the sealing of 5Ј-phosphate and 3Ј-hydroxyl termini at nicks in duplex DNA by means of three distinct catalytic events (1). The first step involves activation of the ligase through the formation of a covalent adenylated intermediate by transfer of the adenyl group of NAD ϩ or ATP to the ⑀-NH 2 of a conserved lysine residue in the DNA ligase. In the second step the AMP moiety is transferred from the DNA ligase to the 5Ј-phosphate group at the single-strand break site, creating a new pyrophosphate bond. Finally, the phosphodiester bond formation is achieved upon an attack of the 3Ј-OH group of the DNA on the activated 5Ј-group with the concomitant release of AMP (1).At least one NAD ϩ -dependent DNA ligase (referred to as LigA) is found in every bacterial species (2). The bacterial LigA enzymes are of fairly uniform size (ϳ70 kDa) and display extensive amino acid sequence conservation throughout the entire protein (3-4). The atomic structures of the LigA enzymes from Bacillus stearothermophilus (N-terminal domain) (5) and Thermus filiformis (3) have been determined by x-ray crystallography. The catalytic core of the bacterial NAD ϩ -dependent DNA ligase con...