Structural and Functional Adaptations to Extreme Temperatures in Psychrophilic, Mesophilic, and Thermophilic DNA Ligases

Abstract

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...

Cofactor Binding Modulates the Conformational Stabilities and Unfolding Patterns of NAD+-dependent DNA Ligases from Escherichia coli and Thermus scotoductus

“…Excitation at 295 nm did not dramatically modify the shape of emission spectra except that the shoulder in the tyrosine emission region (vicinity of 306 nm) disappeared (not shown). The max values recorded for native enzymes indicate that tryptophan (Trp) residues in Eclig and Tslig are rather buried, which is consistent with the localization of Trp residues deduced from three-dimensional modeling studies (14). Furthermore, the adenylation does not change significantly the polarity of Trp environment within the "catalytic core" of the protein (Table II).…”

“…The pET-EcoLIG plasmid (13) was digested with NdeI and BamHI, and the insert was cloned into pET23a plasmid, leading to the overexpression of an untagged protein. The recombinant Eclig was overexpressed and purified as previously described (14). Plasmid encoding Tslig was a kind gift from Z. O. Jónsson and G. Eggertsson (15).…”

“…Plasmid encoding Tslig was a kind gift from Z. O. Jónsson and G. Eggertsson (15). The recombinant wild-type Tslig was overexpressed and purified as previously described (14). Protein concentration was determined with the Coomassie protein assay reagent (Pierce) using bovine serum albumin as standard.…”

“…Compared with Eclig, the unfolding pattern of Tslig is more complex because it is composed of two distinct absorption peaks. This phenomenon is likely to reflect temperature adaptation and has been attributed to a decrease of unfolding cooperativity in the thermophilic enzyme (14). Deadenylated and adenylated Eclig and Tslig denature according to a non-two-state process since deconvolution of the excess heat capacity (C p ) functions reveals that these enzymes possess domains of distinct stability (Table I).…”

“…However, upon adenylation the intrinsic fluorescence is quenched by, respectively, ϳ10 and ϳ35% for Eclig and Tslig (see I max in Table II). Based on three-dimensional modeling studies (14), it is suggested that the observed adenylationinduced fluorescence quenching in Tslig is due to the interaction of AMP with a conserved Tyr-226 contacting the nucleotide and/or Trp-246 and/or Trp-298 located within the active site. This assumption is also relevant for Eclig, since these residues are conserved and located at the same positions in the mesophilic and thermophilic enzymes (14).…”