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 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 not flexibility, in that with the active which is a Biochem. Mol. 2001; PubMed Scopus Google Scholar) that while a psychrophilic and a mesophilic flexibility at °C, the flexibility of the psychrophile was at In to the of data the flexibility of cold-adapted enzymes, on the of and is also and the few are Indeed, Siddiqui K.S. R. T. Extremophiles. 2002; 6: PubMed Scopus Google Scholar) that the of psychrophilic enzymes is due to while M.A. V. G. Biochim. Biophys. Acta. 2000; PubMed Scopus Google Scholar, T. Meuwis M.A. Gerday C. Feller G. J. Mol. Biol. PubMed Scopus Google Scholar, S. Gerday C. Feller G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) found this to be are thus to an or the irreversible and of these enzymes. In to catalytic activity, and conformational flexibility are in psychrophilic enzymes, have investigated DNA The psychrophilic DNA ligase from the Antarctic P. DNA E. DNA T. DNA has been and characterized D. F. Chessa J. U. Gerday C. J. Biochem. 2000; PubMed Scopus Google Scholar), an increased catalytic efficiency as well as an increased of the adaptation of is believed to be due to a decreased of and residues, as well as an overall destabilization of its D. F. Chessa J. U. Gerday C. J. Biochem. 2000; PubMed Scopus Google Scholar). The mesophilic is the DNA ligase V. S. PubMed Scopus Google Scholar) and the thermophilic is the DNA ligase G. A. PubMed Scopus Google Scholar). The enzymes are of a all the properties common to DNA are to of the temperature D. F. Chessa J. U. Gerday C. J. Biochem. 2000; PubMed Scopus Google Scholar), an of enzymes for temperature adaptation In the the overall destabilization of is with the of and of In addition, the relationship between activity, flexibility and stability in is by of its temperature activity, conformational flexibility, and thermal and chemical with its mesophilic and thermophilic and for are the DNA ligases from P. E. and T. The for the proteins was the ligase of T. C. H.K. J. H.K. J. 2000; PubMed Scopus Google Scholar). The and molecular were V. F. S. E. C. V. and G. The temperatures as the were and for P. E. and T. and cold-adapted DNA ligase was at in E. as D. F. Chessa J. U. Gerday C. J. Biochem. 2000; PubMed Scopus Google Scholar), that the was in and that was when The cold enzyme was by by to protein from the and as D. F. Chessa J. U. Gerday C. J. Biochem. 2000; PubMed Scopus Google Scholar). E. DNA ligase was a from V. and S. V. S. PubMed Scopus Google Scholar). The V. S. PubMed Scopus Google Scholar) was with and and the to the of an The was at in E. as V. S. PubMed Scopus Google Scholar), that the was in and that was when The mesophilic protein was to a for T. DNA ligase G. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar), that the were by a was a from and G. G. A. PubMed Scopus Google Scholar). The was and by and by and as G. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). was with the as The was and of for and the of activity was a as D. F. Chessa J. U. Gerday C. J. Biochem. 2000; PubMed Scopus Google Scholar). order to DNA ligases in a enzyme were by an of J. Mol. Biol. PubMed Scopus Google Scholar). The was The were at and for and on were appropriate and were recorded on an Thermal denaturation of the DNA ligases was by the at a protein of in with at and at were the and as PubMed Scopus Google Scholar) and were to a M. G. P. Biochem. J. 1997; PubMed Scopus Google Scholar). were at for a protein of in with at and recorded from to The were for the of The was for Thermal of was at a of In this case, the recorded were for the of at the of conformational of and were characterized by were in and the protein were to an at the than of a were to protein in order to increase by were on an at and °C, at with at and were and and the were recorded for that are the of were for the and for the by for data were as the of in the of to the in the of The data were to to the relation in is the and the J. of Google Scholar). were a as G. D. Gerday C. Biochemistry. PubMed Scopus Google Scholar). were In order to a was to T. 1: Full Text Full Text PDF PubMed Scopus Google Scholar), to a of for and and for was with the and was for were to a in which and of are the The and of the in the and have been A. Eng. PubMed Scopus Google Scholar). on a of the in the were found to be on and on were found to be irreversible the experimental for these was recorded at rates and for and and for and the was from the relation in J.L. M. P.L. Biochemistry. PubMed Scopus Google Scholar), represents the the at a the of the and the evolved at the protein in were at for the cold-adapted in and was by The was to a the and the was from PubMed Scopus Google Scholar) a from was on an at °C, for at an of and at an of of the DNA ligases and was since they denaturation in data were the and as PubMed Scopus Google Scholar). The obtained were a M. G. P. Biochem. J. 1997; PubMed Scopus Google Scholar). of of were as T. Gerday C. Feller G. Biochim. Biophys. Acta. 2000; PubMed Scopus Google Scholar), is the the the at temperature the energy of the and the of and of and show and with T. DNA allowing to from the structure The of and be a of the active of the and of the overall These enzymes are DNA ligases C. H.K. J. H.K. J. 2000; PubMed Scopus Google Scholar) and i.e. and and and or for DNA ligase C. H.K. J. H.K. J. 2000; PubMed Scopus Google Scholar), the of the leads to a large to a that the psychrophilic DNA ligase all the that have been in DNA ligases Moreover, the of the structure of C. H.K. J. H.K. J. 2000; PubMed Scopus Google Scholar), as well as the of of V. S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) have out implicated in catalysis, that to be among DNA ligases to temperatures. further the active of the psychrophilic the as well as the surface of and of the were The trend is a in the and in the surface of to and This is by a of the surface of and an increase of the a the active of the psychrophilic ligase is characterized by an of and reduced when compared with the of the thermophilic trend is observed when the is in the psychrophilic enzyme an increased of to the whereas the thermophilic DNA ligase an increased surface an increase of the to improved in the thermophilic ligase that are to the enzyme at high temperatures, whereas the of in represents an and surface accessibility of the active of and of the active of of of of accessibility of of of of in a of the surface of and accessibility in a is well that the of as with the on the surface of protein results in a increase of the and a of its J. Mol. Biol. 13: PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). the an increased of to the in the of was investigated in the DNA in no of be observed with the In the case of a is the of some at the surface of the of is recorded for a of in the in were also thermal In the case of a increase of is observed when the which a to a surface of and to an lower than that of the a that the increase in is more when the a and at temperatures, still at a than that of the suggests that is more to at high temperatures. are still able to to the observed at high temperatures. The high stability of (see not thermal of of activity of and is in be that the activity of is low temperatures for its weak stability. of the DNA substrate at high temperatures not the of the temperature for activity, results indicate that this is than This a between activity and thermal be in that reaction rates at low and moderate temperatures as compared with and of the temperature of activity and thermal recorded by also an of Indeed, the activity of to the of the explaining the of activity at temperatures. the activity of is and the enzyme is at the of the This suggests an active or catalytic in the cold-adapted conformational flexibility of the DNA ligases was by as the for and at and and a accessibility of in as the temperature of the be compared if the and of in the are as well as their This is not for the DNA ligases and the of the with temperature the Indeed, as in is whereas and are and to the an This is whereas is buried and and are to the and are whereas and are to the the for the The observed for is of a of temperature on the of the protein This a of this enzyme to in a temperature range the Thermal of DNA of and was by was to (see in order to T. 1: Full Text Full Text PDF PubMed Scopus Google Scholar) that the and the range of the DNA ligase and the with thermal recorded by by the large in temperature the psychrophilic enzyme is by the stable when compared with its mesophilic and In addition, the the is recorded for its weak of the and for and and out the in the enzymes of an increased of of stability in the order psychrophile also to a of of of and to the temperature at the of the is the of the to the of by of to the temperature at the of the to the of by of is the of the in a The of on of that thermal of the enzymes is The from a was further by the that the for the enzymes The irreversible conformational of DNA ligases is also by the of their that the thermal denaturation of these proteins is of the of for the denaturation and as the denaturation is for and the energy is and are all for the that the lower of the cold-adapted DNA ligase is due to an The complex of analysis of the for the irreversible in a of DNA stability of the and was further investigated by by in these enzymes to a a stable between the and the enzymes, at with a direct relation between the and the thermal stability. of the of the i.e. stability in the of of the the and of at the with and the psychrophilic the and its weak stability. In addition, the cold-adapted enzyme the the chemical of of and as obtained from the analysis of the are from data in were to Ref. were to Ref. M. G. P. Biochem. J. 1997; PubMed Scopus Google in a and of the DNA ligases and for the of D. F. Chessa J. U. Gerday C. J. Biochem. 2000; PubMed Scopus Google Scholar) and further evidence for the structural implicated in the adaptation to low temperatures of results suggest that the active of the is by an of and a decreased of compared with its thermophilic These are in with the for enzymes D. F. Chessa J. U. Gerday C. J. Biochem. 2000; PubMed Scopus Google Scholar). Indeed, is believed to be the most for for the of low temperatures on the catalytic while for the is to be for which is to at high temperatures. In the the increase of the active is to the with the DNA to an improved of the of the surface of the also out an increase of the surface in the order psychrophile increase to an increase of the at the surface of that to its increased stability. Indeed, in of thermophilic enzymes are believed to a in the adaptation of these enzymes at high temperatures S. Nussinov R. 2001; PubMed Scopus Google Scholar, S. R. Biochemistry. 2002; 41: PubMed Scopus Google Scholar). of the of also a increase of the of of to which is to the protein structure D. F. Chessa J. U. Gerday C. J. Biochem. 2000; PubMed Scopus Google Scholar). While the of the psychrophilic enzyme this it was demonstrated by Indeed, most proteins not since their is well from the by the rigid of J. Mol. Biol. 13: PubMed Scopus Google Scholar). when proteins to the a for the is observed J. Mol. Biol. 13: PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). In the case of the of of the to of The latter are to to an destabilization of the cold-adapted at activity results out that the cold-adapted is characterized by a of the activity low temperatures and an increased specific activity at low and moderate temperatures compared with its mesophilic and thermophilic counterparts. increase of activity to be due to a of the and of the active The for a active in is by the of the of the activity and denaturation of and In the the active is even more than the protein since the activity is conformational change. In the of activity of with that of structural that structural is a for the of activity at high temperatures. is with the rigidity of a whereas at the opposite of the temperature is to be characterized by 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 Scholar, Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar). 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 would be responsible for the weak thermal stability of these enzymes. The flexibility still to since some not this (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, Biochem. Mol. 2001; PubMed Scopus Google Scholar, G. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar, J. J. Biophys. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). in cases, the suffered from the disadvantage of being a measure of the flexibility of the proteins while it is that the increased flexibility of cold-adapted enzymes is and on the to i.e. to the active and catalytic flexibility of and its mesophilic and thermophilic was by as In this the of from between the and the the of the to of the protein the active and be as an of protein J. of Google Scholar). In addition, it conformational a large to as it is a on the DNA ligases indicate that is more flexible than and in a temperature range the flexibility to the high activity in the low temperature also leads to a reduced stability of the molecular common of all psychrophilic enzymes is their low stability in with their mesophilic This has been demonstrated by the of their temperature of activity, the low of the protein to and the high of the structure to at moderate temperatures. The decreased stability of psychrophilic enzymes, in to their increased low temperature activity, suggests that there is a direct between activity and stability i.e. of activity at low temperatures the of which results in reduced stability. The lower stability of psychrophilic enzymes, by a of to the of the protein may from a reduction in of or from in or a few of the structure of the thermal stability of by that this enzyme a molecular that is and by weak than its mesophilic and thermophilic is that the as well as the recorded for cold-adapted enzymes, its psychrophilic through a low conformational stability. decreased stability is also by in which is characterized by the and advances in the of the activity/stability relationship be from the analysis of irreversible thermal of the cold-adapted DNA ligase and its mesophilic results show an increase in of from to that the denaturation is at temperatures (see also The in however from large in the and Indeed, the low recorded for to the and and for trend has been for a few psychrophilic enzymes T. Meuwis M.A. Gerday C. Feller G. J. Mol. Biol. PubMed Scopus Google Scholar, S. Gerday C. Feller G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and thus to be a common of psychrophilic enzymes. The low recorded for the psychrophilic enzyme in at a high to a and denaturation in and to high energy and high This a high of for the psychrophilic that from the lower of to the active The large in suggests that its is more than that of its mesophilic counterpart, due to the that at specific more are the The entropy with of denaturation also to the in between and Indeed, in the case of the mesophilic enzyme and more in the thermophilic counterpart, an increased of are the order of and the entropy to results those obtained for the dehydrogenase M.A. V. G. Biochim. Biophys. Acta. 2000; PubMed Scopus Google Scholar), a cold-adapted S. Gerday C. Feller G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and T. Meuwis M.A. Gerday C. Feller G. J. Mol. Biol. PubMed Scopus Google Scholar), that of psychrophilic enzymes is The obtained by Siddiqui K.S. R. T. Extremophiles. 2002; 6: PubMed Scopus Google Scholar) with the from a archae is to from an of the cold enzyme with a lower than that of the thermophilic the activity/flexibility/stability relationship psychrophilic enzymes was still with the results obtained for the DNA ligases to thermal a between activity, flexibility, and stability. The cold-adapted DNA ligase is characterized by a high activity at low temperatures, a high flexibility and a low stability especially at the active These results are in with those obtained from on S. Gerday C. Feller G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and T. Meuwis M.A. Gerday C. Feller G. J. Mol. Biol. PubMed Scopus Google Scholar). the emerging picture suggests that psychrophilic enzymes are characterized by increased catalytic efficiency attributed to an increase of the flexibility of appropriate parts of the molecular in order to compensate for the lower thermal energy provided by the low temperature habitat. In return, this flexibility would be responsible for the weak thermal and chemical of cold-adapted enzymes. and R. for their also F. T. S. and M. for and