Psychrophiles, host of permanently cold habitats, display metabolic fluxes comparable to those exhibited by mesophilic organisms at moderate temperatures. These organisms have evolved by producing, among other peculiarities, cold-active enzymes that have the properties to cope with the reduction of chemical reaction rates induced by low temperatures. The emerging picture suggests that these enzymes display a high catalytic efficiency at low temperatures through an improved flexibility of the structural components involved in the catalytic cycle, whereas other protein regions, if not implicated in catalysis, may be even more rigid than their mesophilic counterparts. In return, the increased flexibility leads to a decreased stability of psychrophilic enzymes. In order to gain further advances in the analysis of the activity/flexibility/stability concept, psychrophilic, mesophilic, and thermophilic DNA ligases have been compared by three-dimensional-modeling studies, as well as regards their activity, surface hydrophobicity, structural permeability, conformational stabilities, and irreversible thermal unfolding. These data show that the cold-adapted DNA ligase is characterized by an increased activity at low and moderate temperatures, an overall destabilization of the molecular edifice, especially at the active site, and a high conformational flexibility. The opposite trend is observed in the mesophilic and thermophilic counterparts, the latter being characterized by a reduced low temperature activity, high stability and reduced flexibility. These results strongly suggest a complex relationship between activity, flexibility and stability. In addition, they also indicate that in coldadapted enzymes, the driving force for denaturation is a large entropy change.The temperature range in which biological activity has been detected extends from Ϫ20°C, the temperature recorded in the brine veins of Arctic or Antarctic sea ice (1), to 113°C, the temperature at which the archae Pyrolobus fumarii is still able to grow (2). Although numerous investigations have been carried out on thermophilic microorganisms and on their molecular components, especially enzymes, the efforts devoted to coldadapted microorganisms have been comparatively limited despite their tremendous biotechnological (1, 3-5) and fundamental (1, 6 -8) applications. Indeed, the biochemical and physiological bases of cold adaptation, which include, for example, regulation of gene expression by low temperatures, membrane adaptation in relation with the homeoviscosity concept, and the activity/stability relationships sustaining the catalytic efficiency of cold-adapted enzymes, are still poorly understood.In permanent cold habitats, low temperatures have constrained psychrophiles to develop among other peculiarities enzymatic tools allowing metabolic rates compatible to life that are close to those of temperate organisms. Thermal compensation in these enzymes is reached, in most cases, through a high catalytic efficiency at low and moderate temperatures (for revie...

In the last few years, an increased attention has been focused on NAD(+)-dependent DNA ligases. This is mostly due to their potential use as antibiotic targets, because effective inhibition of these essential enzymes would result in the death of the bacterium. However, development of an efficient drug requires that the conformational modifications involved in the catalysis of NAD(+)-dependent DNA ligases are understood. From this perspective, we have investigated the conformational changes occurring in the thermophilic Thermus scotoductus NAD(+)-DNA ligase upon adenylation, as well as the effect of cofactor binding on protein resistance to thermal and chemical (guanidine hydrochloride) denaturation. Our results indicate that cofactor binding induces conformational rearrangement within the active site and promotes a compaction of the enzyme. These data support an induced "open-closure" process upon adenylation, leading to the formation of the catalytically active enzyme that is able to bind DNA. These conformational changes are likely to be associated with the protein function, preventing the formation of nonproductive complexes between deadenylated ligases and DNA. In addition, enzyme adenylation significantly increases resistance of the protein to thermal denaturation and GdmCl-induced unfolding, establishing a thermodynamic link between ligand binding and increased conformational stability. Finally, chemical unfolding of deadenylated and adenylated enzyme is accompanied by accumulation of at least two equilibrium intermediates, the molten globule and premolten globule states. Maximal populations of these intermediates are shifted toward higher GdmCl concentrations in the case of the adenylated ligase. These data provide further insights into the properties of partially folded intermediates.

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...