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• W2010159203 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 cold-adapted enzymes, the driving force for denaturation is a large entropy change. 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 cold-adapted enzymes, the driving force for denaturation is a large entropy change. The temperature range in which biological activity has been detected extends from –20 °C, the temperature recorded in the brine veins of Arctic or Antarctic sea ice (1Deming J.W. Curr. Opin. Microbiol. 2002; 5: 301-309Crossref PubMed Scopus (279) Google Scholar), to 113 °C, the temperature at which the archae Pyrolobus fumarii is still able to grow (2Blochl E. Rachel R. Burggraf S. Hafenbradl D. Jannasch H.W. Stetter K.O. Extremophiles. 1997; 1: 14-21Crossref PubMed Scopus (381) Google Scholar). Although numerous investigations have been carried out on thermophilic microorganisms and on their molecular components, especially enzymes, the efforts devoted to cold-adapted microorganisms have been comparatively limited despite their tremendous biotechnological (1Deming J.W. Curr. Opin. Microbiol. 2002; 5: 301-309Crossref PubMed Scopus (279) Google Scholar, 3Gerday C. Aittaleb M. Arpigny J.L. Baise E. Chessa J.P. Garsoux G. Petrescu I. Feller G. Biochim. Biophys. Acta. 1997; 1342: 119-131Crossref PubMed Scopus (268) Google Scholar, 4Russell N.J. Adv. Biochem. Eng. Biotechnol. 1998; 61: 1-21PubMed Google Scholar, 5Demirjian D.C. Moris-Varas F. Cassidy C.S. Curr. Opin. Chem. Biol. 2001; 5: 144-151Crossref PubMed Scopus (438) Google Scholar) and fundamental (1Deming J.W. Curr. Opin. Microbiol. 2002; 5: 301-309Crossref PubMed Scopus (279) Google Scholar, 6Levy M. Miller S.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7933-7938Crossref PubMed Scopus (238) Google Scholar, 7Wintrode P.L. Miyazaki K. Arnold F.H. J. Biol. Chem. 2000; 275: 31635-31640Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 8Kumar S. Tsai C.J. Nussinov R. Biochemistry. 2002; 41: 5359-5374Crossref PubMed Scopus (72) Google Scholar) 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 review, see Ref. 9Smalas A.O. Leiros H.K. Os V. Willassen N.P. Biotechnol. Annu. Rev. 2000; 6: 1-57Crossref PubMed Scopus (191) Google Scholar and 10Cavicchioli R. Siddiqui K.S. Andrews D. Sowers K.R. Curr. Opin. Biotechnol. 2002; 13: 253-261Crossref PubMed Scopus (396) Google Scholar). The emerging picture is that this increased catalytic efficiency is attributed to an increase of the plasticity or flexibility of appropriate parts of the molecular structure in order to compensate for the lower thermal energy provided by the low temperature habitat. This plasticity would enable a good complementarity with the substrate at a low energy cost, thus explaining the high specific activity of psychrophilic enzymes. In return, this flexibility would be responsible for the weak thermal stability of cold-adapted enzymes. This relationship between activity, flexibility and stability constitutes a hot topic and represents a central issue in the adaptation of proteins to various environments. Moreover, it is believed that all proteins evolve through a balanced compromise between these features, i.e. structural rigidity allowing the retention of a specific three-dimensional-conformation at the physiological temperature and in contrast flexibility, allowing the protein to perform its catalytic function. In the context of temperature adaptation of enzymes, it is assumed that high temperatures require stable protein structure and activity, whereas high enzyme activity is mandatory at low temperatures. While the common trait of a low conformational stability in cold-adapted enzymes has been demonstrated (see Ref. 11D'Amico S. Claverie P. Collins T. Georlette D. Gratia E. Hoyoux A. Meuwis M.A. Feller G. Gerday C. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2002; 357: 917-925Crossref PubMed Scopus (211) Google Scholar for review), the stability/flexibility relationship is still controversial since some authors consider that the instability of psychrophilic enzymes is due to a random genetic drift (12Miyazaki K. Wintrode P.L. Grayling R.A. Rubingh D.N. Arnold F.H. J. Mol. Biol. 2000; 297: 1015-1026Crossref PubMed Scopus (214) Google Scholar). Moreover, if the decreased stability of cold-adapted enzymes is well documented, there is however, no direct experimental evidence of an increased flexibility. Besides, controversial results were obtained when the flexibility of a few psychrophilic enzymes was investigated by measuring hydrogen-deuterieum exchange rates. In the case of 3-isopropylmalate dehydrogenase (13Svingor A. Kardos J. Hajdu I. Nemeth A. Zavodszky P. J. Biol. Chem. 2001; 276: 28121-28125Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), while the psychrophilic and mesophilic enzymes were found more flexible than the thermophilic counterpart, the psychrophile was however more rigid than the mesophile. Nevertheless, in this case, the technique suffered from the disadvantage of being a measure of the accessibility of deeply buried residues, and thus did not detect local flexibility, in particular that associated with the active site which is generally quite accessible. Using a similar technique, Fields (14Fields P.A. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2001; 129: 417-431Crossref PubMed Scopus (347) Google Scholar) showed that while a psychrophilic and a mesophilic lactate dehydrogenases had similar flexibility at 2 °C, the global flexibility of the psychrophile was significantly larger at 23 °C. In addition to the lack of data about the flexibility of cold-adapted enzymes, information on the thermodynamics of inactivation and unfolding is also missing and the few available reports are controversial. Indeed, Siddiqui et al. (15Siddiqui K.S. Cavicchioli R. Thomas T. Extremophiles. 2002; 6: 143-150Crossref PubMed Scopus (43) Google Scholar) proposed that the thermolability of psychrophilic enzymes is due to enthalpic effects, while others (16Ciardiello M.A. Camardella L. Carratore V. di Prisco G. Biochim. Biophys. Acta. 2000; 1543: 11-23Crossref PubMed Scopus (33) Google Scholar, 17Collins T. Meuwis M.A. Gerday C. Feller G. J. Mol. Biol. 2003; 328: 419-428Crossref PubMed Scopus (126) Google Scholar, 18D'Amico S. Gerday C. Feller G. J. Biol. Chem. 2003; 278: 7891-7896Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar) found this to be entropically driven. Further studies are thus required to resolve whether an unfavorable entropic or enthalpic contribution determines the irreversible unfolding and inactivation of these enzymes. In attempt to elucidate how stability, catalytic activity, and conformational flexibility are connected in psychrophilic enzymes, we have investigated three structurally homologous NAD+-dependent DNA ligases. The psychrophilic DNA ligase from the Antarctic bacterium Pseudoalteromonas haloplanktis (Phlig) 1The abbreviations used are: Phlig; P. haloplanktis DNA ligase; FI, fluorescence intensity; DSC, differential scanning calorimetry; Eclig, E. coli DNA ligase; Tslig, T. scotoductus DNA ligase; ANS, 8-anilino-1-naphthalene sulfonic acid; MOPS, 4-morpholinepropanesulfonic acid. has been overexpressed and characterized (19Georlette D. Jonsson Z.O. Van Petegem F. Chessa J.-P. Van Beeumen J. Hubscher U. Gerday C. Eur. J. Biochem. 2000; 267: 3502-3512Crossref PubMed Scopus (55) Google Scholar), high-lighting an increased catalytic efficiency as well as an increased thermolability of the enzyme. Cold adaptation of Phlig is believed to be due to a decreased level of arginine and proline residues, as well as an overall destabilization of its N-terminal domain (19Georlette D. Jonsson Z.O. Van Petegem F. Chessa J.-P. Van Beeumen J. Hubscher U. Gerday C. Eur. J. Biochem. 2000; 267: 3502-3512Crossref PubMed Scopus (55) Google Scholar). The mesophilic reference chosen is the DNA ligase form Escherichia coli (Eclig) (20Sriskanda V. Schwer B. Ho C.K. Shuman S. Nucleic Acids Res. 1999; 27: 3953-3963Crossref PubMed Scopus (63) Google Scholar) and the thermophilic homologue is the Thermus scotoductus DNA ligase (Tslig) (21Thorbjarnardóttir S.H. Jónsson Z.O. Andresson O.S. Kristjánsson J.K. Eggertsson G. Pálsdóttir A. Gene (Amst.). 1995; 161: 1-6Crossref PubMed Scopus (51) Google Scholar). The three enzymes are of a similar size, share all the properties common to NAD+-dependent DNA ligases, but are adapted to different extremes of the temperature scale (19Georlette D. Jonsson Z.O. Van Petegem F. Chessa J.-P. Van Beeumen J. Hubscher U. Gerday C. Eur. J. Biochem. 2000; 267: 3502-3512Crossref PubMed Scopus (55) Google Scholar), therefore constituting an adequate series of homologous enzymes for temperature adaptation studies. In the present work, the overall destabilization of Phlig is examined with the help of three-dimensional modeling and investigation of solvent-exposed hydrophobic clusters. In addition, the intricate relationship between activity, flexibility and stability in Phlig is analyzed by comparison of its temperature dependent activity, conformational flexibility, and thermal and chemical stabilities with its mesophilic and thermophilic counterpart Eclig and Tslig, respectively. Three-dimensional Modeling—The target sequences for modeling are the NAD+-dependent DNA ligases from P. haloplanktis, E. coli, and T. scotoductus. The best template for the three proteins was 1DGS.A, the homologue NAD+-DNA ligase of T. filiformis (22Lee J.Y. Chang C. Song H.K. Moon J. Yang J.K. Kim H.K. Kwon S.T. Suh S.W. EMBO J. 2000; 19: 1119-1129Crossref PubMed Scopus (155) Google Scholar). The modeling, minimizations and molecular dynamics procedures were described elsewhere. 2D. Georlette, V. Blaise, F. Bouillenne, B. Damien, S. H. Thorbjarnardottir, E. Depiereux, C. Gerday, V. N. Uversky, and G. Feller, submitted manuscript. The equilibrium temperatures chosen, known as the optimal organism temperature, were 277, 310, and 354 K, for P. haloplanktis, E. coli, and T. scotoductus, respectively. Protein Preparation and Assays—The recombinant cold-adapted NAD+-dependent DNA ligase (Phlig) was overexpressed at 18 °C in E. coli BL21(DE3) as previously described (19Georlette D. Jonsson Z.O. Van Petegem F. Chessa J.-P. Van Beeumen J. Hubscher U. Gerday C. Eur. J. Biochem. 2000; 267: 3502-3512Crossref PubMed Scopus (55) Google Scholar), except that the growth was performed in TB medium (12 g/liter tryptone, 24 g/liter yeast extract, 4 ml/liter glycerol, 12.54 g/liter K2HPO4, 2.32 g/liter KH2PO4, pH 7) containing 50 μg ml–1 kanamycin, and that induction was performed when OD600 reached ∼4. The cold His-tagged enzyme was then purified by Ni2+-Hitrap Chelating followed by thrombin digestion, second Ni2+-Hitrap Chelating to separate untagged protein from the undigested ligase, and Mono Q HR 5/5, as described previously (19Georlette D. Jonsson Z.O. Van Petegem F. Chessa J.-P. Van Beeumen J. Hubscher U. Gerday C. Eur. J. Biochem. 2000; 267: 3502-3512Crossref PubMed Scopus (55) Google Scholar). Plasmid encoding E. coli DNA ligase was a generous gift from V. Sriskanda and S. Shuman (20Sriskanda V. Schwer B. Ho C.K. Shuman S. Nucleic Acids Res. 1999; 27: 3953-3963Crossref PubMed Scopus (63) Google Scholar). The pET-EcoLIG plasmid (20Sriskanda V. Schwer B. Ho C.K. Shuman S. Nucleic Acids Res. 1999; 27: 3953-3963Crossref PubMed Scopus (63) Google Scholar) was digested with NdeI and BamHI and the insert cloned into pET23a plasmid, leading to the overexpression of an untagged protein. The recombinant Eclig was overexpressed at 37 °C in E. coli BL21(DE3) as previously described (20Sriskanda V. Schwer B. Ho C.K. Shuman S. Nucleic Acids Res. 1999; 27: 3953-3963Crossref PubMed Scopus (63) Google Scholar), except that the growth was performed in TB-ampicillin medium containing 100 μg ml–1 ampicillin, and that induction was performed when OD600 reached ∼3. The mesophilic protein was purified according to a protocol established for T. scotoductus DNA ligase (Tslig) (23Hernandez G. Jenney Jr., F.E. Adams M.W. LeMaster D.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3166-3170Crossref PubMed Scopus (171) Google Scholar), except that the heatings treatments were replaced by a DEAE-agarose column chromatography. Plasmid encoding Tslig was a kind gift from Z. O. Jónsson and G. Eggertsson (Reykjavik, Iceland) (21Thorbjarnardóttir S.H. Jónsson Z.O. Andresson O.S. Kristjánsson J.K. Eggertsson G. Pálsdóttir A. Gene (Amst.). 1995; 161: 1-6Crossref PubMed Scopus (51) Google Scholar). The recombinant wild-type Tslig was overexpressed and purified by two successive heatings (65 and 80 °C), followed by Hi-Trap Heparin and Mono Q HR chromatographic steps, as described (23Hernandez G. Jenney Jr., F.E. Adams M.W. LeMaster D.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3166-3170Crossref PubMed Scopus (171) Google Scholar). Protein concentration was determined with the Coomassie Protein Assay Reagent (Pierce), using bovine serum albumin as standard. The final yield was ∼100 mg, ∼250 mg, and ∼55 mg per liter of culture for Phlig, Eclig, and Tslig respectively. N-terminal sequencing confirmed the integrity of recombinant proteins. Enzyme activity was determined using a fluorimetric assay, as described previously (19Georlette D. Jonsson Z.O. Van Petegem F. Chessa J.-P. Van Beeumen J. Hubscher U. Gerday C. Eur. J. Biochem. 2000; 267: 3502-3512Crossref PubMed Scopus (55) Google Scholar). Adenylation Assays—In order to compare DNA ligases in a similar adenylation state, enzyme stock solutions were adenylated by adding an excess of β-NAD+ (24Timson D.J. Wigley D.B. J. Mol. Biol. 1999; 285: 73-83Crossref PubMed Scopus (66) Google Scholar). The ratio [NAD+]/[DNA ligase] was ∼80. The adenylated mixtures were then incubated at 10 °C (Phlig), 25 °C (Eclig), and 65 °C (Tslig) for 30 min and then cooled rapidly on ice. Protein solutions were dialyzed against appropriate buffer before experiments. Fluorescence Measurements—Both intrinsic and ANS fluorescence measurements were recorded on an Aminco SLM 8100 spectrofluorimeter. Thermal denaturation (scan rate 1 °C/min) of the DNA ligases was monitored by recording the fluorescence intensity change at 330 nm, using a protein concentration of 50 μg/ml (0.66 μm) in 20 mm phosphate sodium, 50 mm NaCl, pH 7.6, with excitation at 280 nm (2-nm band pass) and emission at 330 nm (4-nm band pass). Data were normalized using the pre- and post-transition baseline slopes as described (25Pace C.N. Methods Enzymol. 1986; 131: 266-280Crossref PubMed Scopus (2420) Google Scholar) and were fit according to a three-state model (26Vanhove M. Guillaume G. Ledent P. Richards J.H. Pain R.H. Frere J.M. Biochem. J. 1997; 321: 413-417Crossref PubMed Scopus (34) Google Scholar). ANS fluorescence measurements were performed at 25 °C except for Phlig (15 °C), using a protein concentration of 25 μg/ml (0.33 μm) in 20 mm phosphate sodium, 50 mm NaCl, pH 7.6, with excitation at 390 nm (2-nm band pass), and emission (4-nm band pass) spectra recorded from 420 to 600 nm (scan speed 350 nm min–1). The fluorescence spectra were corrected for the background fluorescence of ANS. The ratio [ANS]/[ligase] was ∼200 using ϵ350nm = 4950 m–1 cm–1 for ANS. Thermal unfolding of samples containing ANS was performed at a scan rate of 1 °C/min. In this case, the recorded fluorescence spectra were corrected for the background fluorescence of ANS at the corresponding temperature. Dynamic Quenching of Fluorescence—The conformational resiliences of Phlig, Eclig, and Tslig were characterized by acrylamide-induced fluorescence quenching. Samples were prepared in 20 mm sodium phosphate buffer, 50 mm NaCl, pH 7.6, and the protein concentrations (∼50 μg/ml) were adjusted to provide an optical density at the excitation wavelength less than 0.1. Aliquots of a 1.2 m acrylamide stock solution were consecutively added to 1 ml protein solution in order to increase acrylamide concentration by ∼5 mm steps. Experiments were performed on an Aminco SLM 8100 spectrofluorimeter at 10 and 25 °C, using excitation at 280 nm with fluorescence emission set at 330 nm (excitation and emission slit widths were 1 and 4, respectively) and the fluorescence intensities were recorded for 30 s. Data, that are the mean of three experiments, were corrected for the dilution effects and for the absorptive screening caused by acrylamide (ϵ280nm = 4.3 m–1 cm–1 for acrylamide). Quenching data were plotted as the ratio of fluorescence in the absence of quencher (F0) to the intensity in the presence of quencher (F) against quencher concentration. The resulting data were fit to dynamic parameters according to the Stern-Volmer relation shown in Equation 1, F0/F=1+KSV[Q](Eq. 1) where KSV is the Stern-Volmer quenching constant and [Q] the quencher concentration (27Lakowicz J. Principles of Fluorescence Spectroscopy. Plenum Press, New York1983: 257-301Crossref Google Scholar). Differential Scanning Calorimetry (DSC)—Measurements were performed using a MicroCal MCS-DSC instrument as detailed (28Feller G. d'Amico D. Gerday C. Biochemistry. 1999; 38: 4613-4619Crossref PubMed Scopus (148) Google Scholar). Samples were dialyzed overnight against 30 mm MOPS, 50 mm KCl, pH 7.6. In order to decrease aggregation, a non-detergent sulfobetaine (3-(1-pyridinio)-1-propane sulfonate) was added prior to DSC experiment (29Goldberg M.E. Expert-Bezançon N. Vuillard L. Rabilloud T. Fold. Des. 1995; 1: 21-27Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), to a final concentration of 0.5 m for Phlig and Eclig, and 0.75 m for Tslig. Protein concentration was determined with the Coomassie Protein Assay Reagent (Pierce), and was ∼4 mg/ml except for Phlig (∼7 mg/ml). Thermograms were analyzed according to a non-two-state model in which Tm , ΔHcal , and ΔHeff of individual transitions are fitted independently using the MicroCal Origin software (version 2.9). The magnitude and source of the errors in the Tm and enthalpies values have been discussed elsewhere (30Matouschek A. Matthews J.M. Johnson C.M. Fersht A.R. Protein Eng. 1994; 7: 1089-1095Crossref PubMed Scopus (95) Google Scholar). Fitting standard errors on a series of three DSC measurements made under the same conditions in the present study were found to be ± 0.1 K on Tm and ±4% on both enthalpies. All scans were found to be irreversible under the experimental conditions used for these studies. Kinetically driven unfolding was recorded at two different scan rates (0.4 and 1 K min–1 for Phlig, and 1 and 2 K min–1 for Eclig), and the rate constant kdenat was calculated from the relation shown in Equation 2 (31Sanchez-Ruiz J.M. Lopez-Lacomba J.L. Cortijo M. Mateo P.L. Biochemistry. 1988; 27: 1648-1652Crossref PubMed Scopus (455) Google Scholar), k=vCp/(ΔHcal-Q)(Eq. 2) where v represents the scan rate (K min–1), Cp the excess heat capacity at a given temperature, ΔHcal the total heat of the process, and Q the heat evolved at the given temperature. GdmCl-induced Unfolding—Approximately 25 μg/ml (A 280nm < 0.1) protein samples in 50 mm phosphate sodium, 50 mm NaCl, pH 7.6, were incubated overnight at 25 °C (18 °C for the cold-adapted Phlig), in defined guanidine hydrochoride (GdmCl) concentrations, and unfolding was monitored by intrinsic fluorescence analysis. The pH was checked to ensure a constant value throughout the whole transition, and the denaturant concentration was determined from refractive index measurements (25Pace C.N. Methods Enzymol. 1986; 131: 266-280Crossref PubMed Scopus (2420) Google Scholar) using a R5000 hand refractometer from Atago. Relative fluorescence was measured on an Aminco SLM 8100 spectrofluorimeter at 25 °C, except for Phlig (18 °C), at an excitation wavelength of 280 nm (2-nm band pass) and at an emission wavelength of 330 nm (4-nm band pass), except Tslig (λ em 333 nm). Unfolding of the three DNA ligases Phlig, Eclig, and Tslig was fully reversible since they regained native conformation following renaturation after complete denaturation in 7 m GdmCl. Fluorescence spectroscopy data were normalized using the pre- and post-transition baseline slopes as described (25Pace C.N. Methods Enzymol. 1986; 131: 266-280Crossref PubMed Scopus (2420) Google Scholar). The transition curves obtained were analyzed using a three-state model (N↔I↔U) (26Vanhove M. Guillaume G. Ledent P. Richards J.H. Pain R.H. Frere J.M. Biochem. J. 1997; 321: 413-417Crossref PubMed Scopus (34) Google Scholar). Determination of Activation Parameters—Thermodynamic parameters of activation were calculated as described (32Lonhienne T. Gerday C. Feller G. Biochim. Biophys. Acta. 2000; 1543: 1-10Crossref PubMed Scopus (294) Google Scholar), using Equations 3, 4, 5, ΔG#=RT×lnkBTh-lnk(Eq. 3) ΔH#=Ea-RT(Eq. 4) ΔS#=(ΔH#-ΔG#)/T(Eq. 5) where kB is the Boltzmann constant (1.3805 10–23 J K–1), h the Planck constant (6.6256 10–34 J s), k (s–1) the rate constant at temperature T (K), Ea the activation energy of the reaction, and R (8.314 J mol–1 K–1) the gas constant. Molecular Model of Adenylated Phlig, Eclig, and Tslig—The amino acid sequences of Phlig, Eclig, and Tslig show 45, 45, and 87% identity with T. filiformis (Tflig) DNA ligase, allowing us to build three-dimensional models from the known x-ray structure (Fig. 1, B–D). The final models of Phlig, Eclig, and Tslig can be superimposed, showing a strong conservation of the active site architecture, of the main secondary structures, and of the overall fold. These monomeric enzymes are folded into four discrete domains characterizing NAD+-dependent DNA ligases (22Lee J.Y. Chang C. Song H.K. Moon J. Yang J.K. Kim H.K. Kwon S.T. Suh S.W. EMBO J. 2000; 19: 1119-1129Crossref PubMed Scopus (155) Google Scholar) (Fig. 1, A and E), i.e. Domain 1 (containing Ia and adenylation subdomains), Domain 2 (OB-fold domain), Domain 3 (Zn-finger and HhH subdomains), and Domain 4 (BRCT or BRCA C-terminal-related domain). As shown for Tf DNA ligase (22Lee J.Y. Chang C. Song H.K. Moon J. Yang J.K. Kim H.K. Kwon S.T. Suh S.W. EMBO J. 2000; 19: 1119-1129Crossref PubMed Scopus (155) Google Scholar), the circular arrangement of the four domains leads to a hole sufficiently large to hold a double-stranded DNA. Sequence alignment reveals that the psychrophilic DNA ligase contains all the conserved boxes that have previously been described in eubacterial DNA ligases (Fig. 1E). Moreover, the recent determination of the structure of Tflig (22Lee J.Y. Chang C. Song H.K. Moon J. Yang J.K. Kim H.K. Kwon S.T. Suh S.W. EMBO J. 2000; 19: 1119-1129Crossref PubMed Scopus (155) Google Scholar), as well as the investigation of Domain Ia of Eclig (33Sriskanda V. Shuman S. J. Biol. Chem. 2002; 277: 9695-9700Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) have pointed out essential residues implicated in catalysis, that appear to be conserved among NAD+-dependent DNA ligases adapted to different temperatures. To further characterize the active site of the psychrophilic enzyme, the number as well as the surface of positively charged, negatively charged, neutral, and nonpolar amino acids of the adenylation domain were determined (Table I). The main trend is a decrease in the number and mainly in the accessible surface of neutral side chains form Phlig to Eclig and Tslig. This is accompanied by a decrease of the accessible surface of nonpolar side chains and an increase of the accessible charged surfaces. As a result, the active site of the psychrophilic ligase is characterized by an excess of hydrophobic surfaces and reduced charged surfaces when compared with the adenylation domain of the thermophilic enzyme. A similar trend is observed when the whole molecule is analyzed (Table II). As shown in Table II, the psychrophilic enzyme displays an increased exposure of hydrophobic residues to the solvent, whereas the thermophilic DNA ligase shows an increased hydrophilic accessible surface area, including an increase of the charged accessible surface. Such discrepancies probably lead to improved electrostatic interactions in the thermophilic ligase that are likely to stabilize the enzyme at high temperatures, whereas the excess of hydrophobic surfaces in Phlig represents an entropy-driven destabilizing factor.Table IComposition and surface accessibility of the active site (adenylation domain) of Phlig, Eclig, and TsligPhligEcligTsligCompositionNumber of amino acids (aa) forming the active site248248246Number of positively charged aa (basic)37 (15%)36 (14%)42 (17%)Number of negatively charged aa (acidic)35 (14%)33 (13%)39 (15%)Number of neutral aa52 (21%)45 (18%)36 (14%)Number of nonpolar aa124 (50%)134 (54%)129 (52%)Surface accessibility (Å2)Recalculated accessible surface143751357513888Surface of positively charged aa (basic)4339 (30%)4329 (31%)5629 (40%)Surface of negatively charged aa (acidic)2579 (18%)2510 (18%)3220 (23%)Surface of neutral aa2911 (20%)2519 (18%)1312 (9%)Surface of nonpolar aa4546 (32%)4217 (31%)3727 (26%) Open table in a new tab Table IIComposition of the accessible surface of Phlig, Eclig, and TsligPhligEcligTsligSurface accessibility (Å2)Recalculated accessible surface361833608536626Neutral accessible surface3254 (9%)2362 (7%)2269 (6%)Hydrophobic accessible surface7994 (22%)6468 (18%)6270 (17%)Hydrophilic accessible surface24935 (69%)27255 (75%)28087 (77%)positively charged9202 (25%)11800 (33%)13129 (36%)negatively charged7184 (20%)9187 (25%)8834 (24%) Open table in a new tab ANS Fluorescence—It is well known that the interaction of hydrophobic fluorescent probes such as 8-anilino-1-naphtalenesulfonic acid (ANS) with the exposed hydrophobic sites on the surface of native protein results in a considerable increase of the dye fluorescence intensity and a blue shift of its fluorescence spectrum (34Stryer L. J. Mol. Biol. 1965; 13: 482-495Crossref PubMed Scopus (1338) Google Scholar, 35Semisotn" @default.
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• W2010159203 title "Structural and Functional Adaptations to Extreme Temperatures in Psychrophilic, Mesophilic, and Thermophilic DNA Ligases" @default.
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