Review Molecular adaptations to cold in psychrophilic enzymes
Review Molecular adaptations to cold in psychrophilic enzymes
Review Molecular adaptations to cold in psychrophilic enzymes
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CMLS, Cell. Mol. Life Sci. 60 (2003) 648–6621420-682X/03/040648-15DOI 10.1007/s00018-003-2155-3© Birkhäuser Verlag, Basel, 2003CMLS Cellular and <strong>Molecular</strong> Life Sciences<strong>Review</strong><strong>Molecular</strong> <strong>adaptations</strong> <strong>to</strong> <strong>cold</strong> <strong>in</strong> <strong>psychrophilic</strong> <strong>enzymes</strong>G. FellerLabora<strong>to</strong>ry of Biochemistry, Institute of Chemistry B6, University of Liège, 4000 Liège (Belgium),Fax +32 4 366 33 64, e-mail: gfeller@ulg.ac.beReceived 21 June 2002; received after revision 9 September 2002; accepted 10 September 2002Abstract. Psychrophiles or <strong>cold</strong>-lov<strong>in</strong>g organisms successfullycolonize <strong>cold</strong> environments of the Earth’s biosphere.To cope with the reduction of chemical reactionrates <strong>in</strong>duced by low temperatures, these organisms synthesize<strong>enzymes</strong> characterized by a high catalytic activityat low temperatures associated, however, with low thermalstability. Thanks <strong>to</strong> recent advances provided by X-ray crystallography, prote<strong>in</strong> eng<strong>in</strong>eer<strong>in</strong>g and biophysicalstudies, we are beg<strong>in</strong>n<strong>in</strong>g <strong>to</strong> understand the molecular<strong>adaptations</strong> responsible for these properties which appear<strong>to</strong> be relatively diverse. The emerg<strong>in</strong>g picture suggeststhat <strong>psychrophilic</strong> <strong>enzymes</strong> utilize an improved flexibilityof the structures <strong>in</strong>volved <strong>in</strong> the catalytic cycle,whereas other prote<strong>in</strong> regions if not implicated <strong>in</strong> catalysismay or may not be subjected <strong>to</strong> genetic drift.Key words. Psychrophile; extremophile; enzyme k<strong>in</strong>etics; crystal structure; fold<strong>in</strong>g; mutagenesis; biophysics.The current contextS<strong>in</strong>ce our last review <strong>in</strong> this journal [1], significant advanceshave been made <strong>in</strong> the study of prote<strong>in</strong>s and <strong>enzymes</strong>from <strong>cold</strong>-adapted organisms, the so-called psychrophiles,demonstrat<strong>in</strong>g an <strong>in</strong>tense research activity <strong>in</strong>the field. Such <strong>in</strong>terest is certa<strong>in</strong>ly motivated by the factthat life at extremely low temperatures is no longer regardedas a biological curiosity. Indeed, the <strong>cold</strong>-activeand heat-labile <strong>psychrophilic</strong> <strong>enzymes</strong> possess <strong>in</strong>terest<strong>in</strong>gbiotechnological potential [2–6] and have beenshown <strong>to</strong> be useful models for prote<strong>in</strong> fold<strong>in</strong>g studies [7]and for the analysis of the adaptability of the structureand function of <strong>enzymes</strong> [8]. In addition, a low-temperatureorig<strong>in</strong> of life is still a plausible hypothesis [9]. Moreexotic reasons for this <strong>in</strong>terest should also be mentioned.For example, liv<strong>in</strong>g remnants of ancestral microorganisms,such as those expected <strong>to</strong> survive below 3500 m ofice <strong>in</strong> the Antarctic subglacial Lake Vos<strong>to</strong>k [10], shoulddisplay <strong>psychrophilic</strong> characters, as should the possibleliv<strong>in</strong>g forms on the planet Mars [11]. Psychrophiles werefirst referred <strong>to</strong> as <strong>cold</strong>-adapted bacteria [12]. However, ifa psychrophile is def<strong>in</strong>ed as an organism liv<strong>in</strong>g permanentlyat temperatures close <strong>to</strong> the freez<strong>in</strong>g po<strong>in</strong>t of water<strong>in</strong> thermal equilibrium with the medium, such a def<strong>in</strong>ition<strong>in</strong>cludes de fac<strong>to</strong> a large range of species: yeast[13–16], algae [17–19], mar<strong>in</strong>e <strong>in</strong>vertebrates and <strong>in</strong>sectsor polar fish, which are the largest psychrophiles [20, 21].These examples underl<strong>in</strong>e that psychrophiles are numerous,taxonomically diverse and have a widespread distribution.One should not forget that the major part of Earthis exposed <strong>to</strong> low temperatures if one considers the vastextent of permanently <strong>cold</strong> environments such as theAntarctic, Arctic and mounta<strong>in</strong> regions as well as thedeep-sea waters cover<strong>in</strong>g three-quarters of the planet surface.To def<strong>in</strong>e the properties of a <strong>cold</strong>-active enzyme, the effec<strong>to</strong>f temperature on the activity of <strong>psychrophilic</strong> andmesophilic <strong>enzymes</strong> is illustrated <strong>in</strong> figure 1. This graphreveals three basic features which will form the body ofthe next sections.1) To compensate for the slow reaction rates at low temperatures,psychrophiles synthesize <strong>enzymes</strong> with an
CMLS, Cell. Mol. Life Sci. Vol. 60, 2003 <strong>Review</strong> Article 649Figure 1. Effect of temperature on the activity of <strong>psychrophilic</strong> ()and mesophilic () <strong>enzymes</strong>. The thermodependence of the activity(of a-amylases <strong>in</strong> this case) illustrates the three ma<strong>in</strong> adaptive traitsof <strong>psychrophilic</strong> <strong>enzymes</strong>.up <strong>to</strong> tenfold higher specific activity <strong>in</strong> this temperaturerange. This is <strong>in</strong> fact the ma<strong>in</strong> physiological adaptation<strong>to</strong> <strong>cold</strong> at the enzyme level.2) The temperature for apparent maximal activity for<strong>cold</strong>-active <strong>enzymes</strong> is shifted <strong>to</strong>wards low temperatures,reflect<strong>in</strong>g the weak stability of these prote<strong>in</strong>swhich are prone <strong>to</strong> unfold<strong>in</strong>g and <strong>in</strong>activation at moderatetemperatures.3) F<strong>in</strong>ally, the adaptation <strong>to</strong> <strong>cold</strong> is frequently not perfect.Figure 1 illustrates that the specific activity of the <strong>psychrophilic</strong>enzyme at low temperatures, although veryhigh, rema<strong>in</strong>s generally lower than that of themesophilic <strong>enzymes</strong> at 37°C.Some reviews have been published recently by our group[22–26] but other excellent reviews should also be consultedfor a complete coverage of this <strong>to</strong>pic [4, 11,27–33].Recomb<strong>in</strong>ant <strong>psychrophilic</strong> <strong>enzymes</strong>A major achievement <strong>in</strong> the study of <strong>psychrophilic</strong> <strong>enzymes</strong>was the availability of recomb<strong>in</strong>ant <strong>enzymes</strong> thathave been produced <strong>in</strong> sufficient amount <strong>to</strong> determ<strong>in</strong>e X-ray structures and for their analysis by sophisticated biophysicalmethods. This has also opened the way for mutagenesisstudies. The first <strong>cold</strong>-active <strong>enzymes</strong> werecloned <strong>in</strong> Escherichia coli and selected by the activity ofthe gene product expressed by the recomb<strong>in</strong>ant colonies[34–36]. Later, the high level of sequence identity withmesophilic homologues demonstrated by <strong>psychrophilic</strong><strong>enzymes</strong> prompted the isolation by the polymerase cha<strong>in</strong>reaction of other genes that cannot be detected <strong>in</strong> expressionlibraries [37, 38]. Accord<strong>in</strong>gly, the study of rare <strong>enzymes</strong>was also made possible, such as the elongationfac<strong>to</strong>rs EF-2 [39–41] and Tu [42] or a DNA ligase [43]which only occurs as 200 molecules per cell, thereforepreclud<strong>in</strong>g its direct purification <strong>in</strong> reasonable amounts.The availability of these genes also allowed an explorationof the overexpression of these <strong>psychrophilic</strong> <strong>enzymes</strong><strong>in</strong> a mesophilic host. Various E. coli stra<strong>in</strong>s wereused for this purpose, whereas expression <strong>in</strong> Bacillusspecies [44, 45] or yeast was rarely addressed. The firstconcern of workers <strong>in</strong> the field was <strong>to</strong> establish if an enzymeexpressed near 0°C by the wild-type stra<strong>in</strong> ma<strong>in</strong>ta<strong>in</strong>sits properties when expressed and folded at highertemperatures that are lethal for the parent microorganisms.Indeed, when expressed at a sufficiently low temperature(15–18°C), a recomb<strong>in</strong>ant <strong>psychrophilic</strong> enzymeis undist<strong>in</strong>guishable from its wild-type parent moleculeas regards k<strong>in</strong>etic parameters, fold<strong>in</strong>g properties[46] and three-dimensional (3D) structure [47]. By contrast,at 37°C, recomb<strong>in</strong>ant <strong>psychrophilic</strong> <strong>enzymes</strong> arefrequently not expressed or are quickly <strong>in</strong>activated. In addition,when expressed at this temperature, the propertiesof the recomb<strong>in</strong>ant <strong>psychrophilic</strong> enzyme seem <strong>to</strong> be alteredas a result of misfold<strong>in</strong>g or the occurrence of an <strong>in</strong>activatedpopulation [46]. Aff<strong>in</strong>ity tags added <strong>in</strong> fusion <strong>to</strong>a recomb<strong>in</strong>ant <strong>psychrophilic</strong> enzyme for purification purposesare also a concern. A His-tagged phosphoglyceratek<strong>in</strong>ase (PGK) has <strong>in</strong>termediate k<strong>in</strong>etic properties betweenthe native <strong>psychrophilic</strong> PGK and yeast PGK, adifference possibly aris<strong>in</strong>g from the stabilization center<strong>in</strong>troduced by the aff<strong>in</strong>ity tag [48]. As a practical conclusion,it is therefore advisable <strong>to</strong> express recomb<strong>in</strong>ant <strong>psychrophilic</strong><strong>enzymes</strong> at the lowest possible temperature, <strong>to</strong>cleave and remove aff<strong>in</strong>ity tags, <strong>to</strong> perform an aff<strong>in</strong>itychroma<strong>to</strong>graphy step (remov<strong>in</strong>g the possible <strong>in</strong>activefraction) and <strong>to</strong> perform active-site titration <strong>to</strong> calculatethe k<strong>in</strong>etic parameters.Recently, a recomb<strong>in</strong>ant <strong>psychrophilic</strong> enzyme has beenexpressed <strong>in</strong> <strong>psychrophilic</strong> bacteria [49]. For this purpose,a broad host range expression vec<strong>to</strong>r was constructedus<strong>in</strong>g a <strong>cold</strong>-adapted replication element derivedfrom a multicopy cryptic plasmid of the Antarctic Gramnegativebacterium Pseudoalteromonas haloplanktis.This study demonstrated that production of recomb<strong>in</strong>antprote<strong>in</strong>s can be performed at low temperatures, reveal<strong>in</strong>ga new biotechnological potential for <strong>psychrophilic</strong>stra<strong>in</strong>s.Structural fac<strong>to</strong>rs related <strong>to</strong> activityat low temperatureTo date, four crystal structures of bacterial <strong>psychrophilic</strong><strong>enzymes</strong> have been solved by X-ray crystallography[50–53] as has the structure of three ser<strong>in</strong>e proteasesfrom <strong>cold</strong>-adapted fish [54–56]. Other structures shouldbecome available soon as crystals of three other <strong>cold</strong>-ac-
650 G. Feller Cold-active <strong>enzymes</strong>tive <strong>enzymes</strong> have been obta<strong>in</strong>ed [57–59]. Unfortunately,only four of these X-ray structures have been comparedwith mesophilic or thermophilic homologues and analyzed<strong>in</strong> detail with regards <strong>to</strong> the structural parametersrelated <strong>to</strong> <strong>cold</strong> activity. These <strong>in</strong>clude the bacterial a-amylase [47], citrate synthase [53], malate dehydrogenase[52] and the fish tryps<strong>in</strong> [54]. Nevertheless, this limitedset of data has already provided valuable <strong>in</strong>sights <strong>in</strong><strong>to</strong>the molecular basis of <strong>cold</strong> adaptation [30, 60–62]. As <strong>in</strong>ferredfrom their am<strong>in</strong>o acid sequences and homologymodels, <strong>psychrophilic</strong> <strong>enzymes</strong> do not display unusual orexotic conformations. On the contrary, the overall fold isvery close <strong>to</strong> that of their mesophilic homologues. For example,when the 3D structures of mammalian a-amylasesare superimposed on the <strong>psychrophilic</strong> a-amylase, theroot mean square deviation is only about 0.5 Å, reflect<strong>in</strong>ga high similarity of the tertiary structures [47].As far as the active site of <strong>psychrophilic</strong> <strong>enzymes</strong> is concerned,all reactive side cha<strong>in</strong>s as well as most side cha<strong>in</strong>spo<strong>in</strong>t<strong>in</strong>g <strong>to</strong>wards the catalytic cavity are strictly conserved.This means that the overall catalytic mechanismand reaction pathway are not modified <strong>in</strong> <strong>cold</strong>-active <strong>enzymes</strong>.This aspect is not really surpris<strong>in</strong>g as the specificreaction mechanism of <strong>enzymes</strong> is not prone <strong>to</strong> drasticvariation. However, worth mention<strong>in</strong>g is that all residuesform<strong>in</strong>g the active site can be conserved <strong>in</strong> a <strong>psychrophilic</strong>enzyme. This was demonstrated by the X-raystructure solved at high resolution of both the <strong>cold</strong>-activea-amylase and of its closest structural homologue frompig <strong>in</strong> complex with acarbose, a pseudosaccharide <strong>in</strong>hibi<strong>to</strong>rmimick<strong>in</strong>g the transition state <strong>in</strong>termediate [63,64]. Comparison of residues with direct or water-mediatedH bonds <strong>to</strong> the <strong>in</strong>hibi<strong>to</strong>r revealed that all 24 residues<strong>in</strong>volved are strictly conserved <strong>in</strong> the <strong>cold</strong>-active a-amylase.This outstand<strong>in</strong>g example of active-site identity (<strong>in</strong>terms of side cha<strong>in</strong> identity) demonstrates that the specificproperties of <strong>psychrophilic</strong> <strong>enzymes</strong> can be reachedwithout any am<strong>in</strong>o acid substitution <strong>in</strong> the reaction center.As a consequence, changes occurr<strong>in</strong>g elsewhere <strong>in</strong> themolecule are responsible for the optimization of the catalyticparameters of this enzyme, as demonstrated by sitedirectedmutagenesis [65], probably by modify<strong>in</strong>g the dynamicand conformational properties of the conservedresidues.Another important feature of the active site refers <strong>to</strong> itsaccessibility, because <strong>in</strong> many cases, the catalytic cavityappears <strong>to</strong> be larger and more accessible <strong>to</strong> ligands. Thislarger open<strong>in</strong>g of the catalytic cleft is achieved <strong>in</strong> variousways, <strong>in</strong>clud<strong>in</strong>g small deletions <strong>in</strong> loops border<strong>in</strong>g the activesite or by dist<strong>in</strong>ct conformation of these loops, and byreplacement of bulky side cha<strong>in</strong>s for smaller groups at theentrance. This aspect has been well illustrated for the<strong>cold</strong>-active citrate synthase [53]. The reasons for such abetter accessibility are still not clearly unders<strong>to</strong>od but<strong>cold</strong>-active <strong>enzymes</strong> are thought <strong>to</strong> accommodate substratesat lower energy cost, as far as the conformationalchanges are concerned, and therefore reduce the activationenergy required for the formation of the enzyme-substratecomplex. The larger active site may also facilitateeasier release and exit of products and thus may alleviatethe effect of a rate-limit<strong>in</strong>g step on the reaction rate. Thislarger size of the active site and the better accessibilityhave at least two consequences. First, non-specific <strong>enzymes</strong>accept<strong>in</strong>g various substrates should have a broaderspecificity, because substrates with slightly dist<strong>in</strong>ct conformationsor sizes can fit and b<strong>in</strong>d <strong>to</strong> the site. For example,the observed differences <strong>in</strong> substrate specificity betweenAtlantic salmon and mammalian elastases havebeen <strong>in</strong>terpreted <strong>in</strong> terms of a somewhat wider and deeperb<strong>in</strong>d<strong>in</strong>g pocket for the <strong>cold</strong>-adapted elastase [28]. Thebroad specificity of a <strong>psychrophilic</strong> alcohol dehydrogenase,oxidiz<strong>in</strong>g large bulky alcohols, was also assigned <strong>to</strong>a highly flexible active site [66]. As a second consequence,substrates should b<strong>in</strong>d less firmly <strong>in</strong> the b<strong>in</strong>d<strong>in</strong>gsite, if no po<strong>in</strong>t mutations have occurred, giv<strong>in</strong>g rise <strong>to</strong>higher K m values. This is certa<strong>in</strong>ly one of the structuralexplanations for the observation that numerous <strong>cold</strong>active<strong>enzymes</strong>, which are strongly homologous <strong>to</strong>mesophilic counterparts, have lower b<strong>in</strong>d<strong>in</strong>g constants.F<strong>in</strong>ally, differences <strong>in</strong> electrostatic potentials <strong>in</strong> andaround the active site of <strong>psychrophilic</strong> <strong>enzymes</strong> appear <strong>to</strong>be a crucial parameter for activity at low temperatures.Interest<strong>in</strong>gly, the <strong>cold</strong>-active citrate synthase [53], malatedehydrogenase [52] and tryps<strong>in</strong> [28, 67, 68] are characterizedby marked differences <strong>in</strong> electrostatic potentialsnear the active-site region when compared with mesophilicor thermophilic counterparts. In the case of malatedehydrogenase for example, the <strong>in</strong>creased positive potentialat and around the oxaloacetate-b<strong>in</strong>d<strong>in</strong>g site and thesignificantly decreased negative surface potential at theNADH-b<strong>in</strong>d<strong>in</strong>g region may facilitate the <strong>in</strong>teraction ofthe oppositely charged ligands with the surface of the enzyme.In all cases, the differences were caused by discretesubstitutions <strong>in</strong> non-conserved charged residues result<strong>in</strong>g<strong>in</strong> local electrostatic potential differ<strong>in</strong>g <strong>in</strong> both sign andmagnitude. Future work <strong>in</strong> the field will certa<strong>in</strong>ly ref<strong>in</strong>ethe structural and functional relationships between electrostaticpotentials and catalytic efficiency <strong>in</strong> <strong>psychrophilic</strong><strong>enzymes</strong>.Structural fac<strong>to</strong>rs l<strong>in</strong>ked <strong>to</strong> stabilityThe numerous homology-based models and especiallythe recently solved X-ray structures of <strong>psychrophilic</strong> <strong>enzymes</strong>have deciphered the molecular orig<strong>in</strong> of the weakstability characteriz<strong>in</strong>g these prote<strong>in</strong>s. All structural fac<strong>to</strong>rscurrently known <strong>to</strong> stabilize the prote<strong>in</strong> molecule canbe attenuated <strong>in</strong> strength and number <strong>in</strong> the structure of<strong>cold</strong>-active <strong>enzymes</strong> [28, 30, 62]. This <strong>in</strong>volves the clus-
CMLS, Cell. Mol. Life Sci. Vol. 60, 2003 <strong>Review</strong> Article 651ter<strong>in</strong>g of glyc<strong>in</strong>e residues (provid<strong>in</strong>g local mobility), thedisappearance of prol<strong>in</strong>e residues <strong>in</strong> loops (provid<strong>in</strong>g enhancedcha<strong>in</strong> flexibility between secondary structures), areduction <strong>in</strong> arg<strong>in</strong><strong>in</strong>e residues which are capable of form<strong>in</strong>gmultiple salt bridges and H bonds, as well as a lowernumber of ion pairs, aromatic <strong>in</strong>teractions or H bondscompared <strong>to</strong> mesophilic <strong>enzymes</strong>. The size and relativehydrophobicity of non-polar residue clusters form<strong>in</strong>g theprote<strong>in</strong> core are frequently smaller, lower<strong>in</strong>g the compactnessof the prote<strong>in</strong> <strong>in</strong>terior. The N and C caps of a-helices are also altered (weaken<strong>in</strong>g the charge-dipole <strong>in</strong>teraction)and loose or relaxed prote<strong>in</strong> extremities appear<strong>to</strong> be preferential sites for unzipp<strong>in</strong>g. The b<strong>in</strong>d<strong>in</strong>g of stabiliz<strong>in</strong>gions, such as calcium, can be extremely weak,with b<strong>in</strong>d<strong>in</strong>g constants differ<strong>in</strong>g from mesophiles by severalorders of magnitude. Insertions and deletions aresometimes responsible for specific properties such as theacquisition of extra surface charges (<strong>in</strong>sertion) or theweaken<strong>in</strong>g of subunit <strong>in</strong>teractions (deletion).Calculation of the solvent-accessible area showed thatsome <strong>psychrophilic</strong> <strong>enzymes</strong> expose a higher proportionof non-polar residues <strong>to</strong> the surround<strong>in</strong>g medium [47,53]. This is an entropy-driven destabiliz<strong>in</strong>g fac<strong>to</strong>r causedby the reorganization of water molecules around exposedhydrophobic side cha<strong>in</strong>s. Calculations of the electrostaticpotential revealed <strong>in</strong> some cases an excess of negativecharges at the surface of the molecule and, <strong>in</strong>deed, the pIof <strong>cold</strong>-active <strong>enzymes</strong> is frequently more acidic than tha<strong>to</strong>f their mesophilic or thermophilic homologues. This hasbeen related <strong>to</strong> improved <strong>in</strong>teractions with the solvent,which could be of prime importance <strong>in</strong> the acquisition offlexibility near zero degrees [69]. Besides the balance ofcharges, the number of salt bridges cover<strong>in</strong>g the prote<strong>in</strong>surface is also reduced. There is now a clear correlationbetween surface ion pairs and temperature adaptation,s<strong>in</strong>ce these weak <strong>in</strong>teractions significantly <strong>in</strong>crease <strong>in</strong>number from psychrophiles <strong>to</strong> mesophiles, thermophilesand hyperthermophiles, the latter show<strong>in</strong>g arg<strong>in</strong><strong>in</strong>e-mediatedmultiple ion pairs and <strong>in</strong>terconnected salt bridgenetworks [70, 71]. Such an altered pattern of electrostatic<strong>in</strong>teractions at the molecular surface is thought <strong>to</strong> improvethe resilience [72] or the ‘breath<strong>in</strong>g’ of the externalshell of <strong>cold</strong>-active <strong>enzymes</strong>.Of course, all these fac<strong>to</strong>rs are not found <strong>in</strong> every <strong>cold</strong>-activeenzyme: each enzyme adopts its own strategy by us<strong>in</strong>gone or a comb<strong>in</strong>ation of these altered structural fac<strong>to</strong>rs<strong>to</strong> improve the local or global mobility of the prote<strong>in</strong>edifice. An example is given <strong>in</strong> table 1 which provides asummary of the structural fac<strong>to</strong>rs possibly <strong>in</strong>volved <strong>in</strong> thelow stability of a <strong>psychrophilic</strong> a-amylase. A recent comparativestructural analysis of <strong>psychrophilic</strong>, mesophilicand thermophilic <strong>enzymes</strong> reports that each prote<strong>in</strong> familydisplays a different structural strategy <strong>to</strong> adapt <strong>to</strong> temperature.However, some common trends are observed:the number of ion pairs, the side cha<strong>in</strong> contribution <strong>to</strong> theTable 1. Structural parameters potentially <strong>in</strong>volved <strong>in</strong> the stabilityof a-amylase conformation as deduced from the X-ray structures[47, 63, 74]a-amylase Psychrophile MesophileProl<strong>in</strong>e content 13 21Salt bridges 18 26Arg<strong>in</strong><strong>in</strong>e content 13 28Arg-mediated salt bridges 8 17Arg-mediated am<strong>in</strong>o-aromatic 2 13<strong>in</strong>teractionsArg-mediated H bonds 20 42Disulfide bonds 4 5Core cluster hydrophobicity (S H i ) 47 103Non-constra<strong>in</strong>ed residues at 6 0C term<strong>in</strong>usAromatic-aromatic <strong>in</strong>teractions 19 20Am<strong>in</strong>o-aromatic <strong>in</strong>teractions 2 13Oxygen-aromatic <strong>in</strong>teractions 24 19Sulfur-aromatic <strong>in</strong>teractions 2 5a helix dipole – charges <strong>in</strong> Na 2 4+ charges <strong>in</strong> Ca 3 6Calcium-b<strong>in</strong>d<strong>in</strong>g constant (M –1 ) 5.0 ¥ 10 7 2.0 ¥ 10 11Chloride-b<strong>in</strong>d<strong>in</strong>g constant (M –1 ) 1.6 ¥ 10 2 3.5 ¥ 10 3Data for the <strong>psychrophilic</strong> a-amylase differ slightly from Aghajariet al. [47] as only one crystal form is considered and bond lengthlimits are different.exposed surface and the apolar fraction of the buried surfaceshow a consistent decrease with decreas<strong>in</strong>g optimaltemperatures [62].K<strong>in</strong>etic optimization of <strong>cold</strong>-active <strong>enzymes</strong>As mentioned, improv<strong>in</strong>g the turnover number k cat is thema<strong>in</strong> physiological adaptation, because it offsets the <strong>in</strong>hibi<strong>to</strong>ryeffect of low temperatures on reaction rates andtherefore provides adequate raw metabolic activity <strong>to</strong> thegrow<strong>in</strong>g organism. However, both k cat and K m are fundamentalk<strong>in</strong>etic parameters characteriz<strong>in</strong>g an enzymaticreaction and the specificity constant k cat /K m is generally abetter <strong>in</strong>dication of catalytic efficiency than k cat alone. Inpr<strong>in</strong>ciple, <strong>cold</strong>-adapted <strong>enzymes</strong> can optimize the k cat /K mratio by <strong>in</strong>creas<strong>in</strong>g k cat , decreas<strong>in</strong>g K m or by changes <strong>in</strong>both k cat and K m . Inspection of the available data, althoughlimited [for compilations see refs 4 and 28], reveals twoma<strong>in</strong> trends: <strong>cold</strong>-active <strong>enzymes</strong> that improve k cat at theexpense of K m (both k cat and K m <strong>in</strong>crease) and those improv<strong>in</strong>gthe k cat /K m ratio (<strong>in</strong>crease of k cat and decrease ofK m ). As will be discussed <strong>in</strong> the next sections, both k<strong>in</strong>eticbehaviors seem <strong>to</strong> be related <strong>to</strong> <strong>cold</strong>-active <strong>enzymes</strong> devoidof adaptive mutations with<strong>in</strong> the active site and <strong>to</strong><strong>enzymes</strong> display<strong>in</strong>g adaptive mutations with<strong>in</strong> the catalyticcenter, respectively.
652 G. Feller Cold-active <strong>enzymes</strong>Improv<strong>in</strong>g the turnover number k cat :a thermodynamic challengeThe catalytic constant k cat corresponds <strong>to</strong> the maximumnumber of substrate molecules converted <strong>to</strong> product peractive site per unit of time. In the simple Michaelis-Menten mechanism, the k cat parameter is the first-orderrate constant for the chemical conversion of the <strong>enzymes</strong>ubstratecomplex <strong>to</strong> enzyme and product. The transitionstate theory assumes the existence of a stable activatedcomplex ES # <strong>in</strong> equilibrium with the ground state ES:k catE + S ´ ES ´ ES# æÆ E + P (1)and the temperature dependence of the catalytic rate constantis given by the follow<strong>in</strong>g relation which is formallyequivalent <strong>to</strong> the Arrhenius law:k B Tk cat = k 7 e – DG# /RT(2)hwhere k is the transmission coefficient generally close <strong>to</strong>1, k B is the Bolzmann constant (1.3805 ¥ 10 –23 J K –1 ), hthe Planck constant (6.6256 ¥ 10 –34 J s) and DG # the freeenergy of activation or the variation of the Gibbs energybetween the activated enzyme-substrate complex ES # andES. The transition state theory conta<strong>in</strong>s several wellknownsimplifications and <strong>in</strong>terpretation of the absolutevalue of the thermodynamic parameters of activationshould be taken with caution. In the context of <strong>cold</strong>adaptation has come the proposal <strong>to</strong> compare the variationof the thermodynamic parameters [25] between <strong>psychrophilic</strong>( p ) and mesophilic ( m ) <strong>enzymes</strong>, namelyD(DG # ) p–m , D(DH # ) p–m and D(DS # ) p–m . Such parametersare compiled <strong>in</strong> table 2. It should be stressed that commonlyfound negative values provide useful <strong>in</strong>sights <strong>in</strong><strong>to</strong>the way <strong>psychrophilic</strong> <strong>enzymes</strong> have evolved.As <strong>in</strong>ferred from equation 2, the D(DG # ) p–m parametersimply reflects the fact that <strong>psychrophilic</strong> <strong>enzymes</strong> aremore active than their mesophilic homologues. However,the small value of this parameter orig<strong>in</strong>ates from largedifferences <strong>in</strong> the enthalpic and entropic contributions,D(DH # ) p–m and TD(DS # ) p–m , respectively, between <strong>cold</strong>adaptedand mesophilic <strong>enzymes</strong>. To appreciate the <strong>in</strong>formativecontent of D(DH # ) p–m , the exponential term ofequation 2 can be rewritten as:k B Tk cat = 7 e – (DH# /RT – DS # /R) (3)hIn this form, one can note that either a decrease of DH # oran <strong>in</strong>crease of DS # results <strong>in</strong> an <strong>in</strong>crease of k cat . However,the contribution of the DH # /RT term <strong>to</strong> the decrease of k catis larger when the temperature T is lowered. Interest<strong>in</strong>gly,all <strong>psychrophilic</strong> <strong>enzymes</strong> studied so far possess lowerTable 2. Activation parameters for the activity of <strong>psychrophilic</strong> and mesophilic <strong>enzymes</strong> [25].Enzyme Source T k cat DG # DH # TDS # D(DG # ) p-m D(DH # ) p-m TD(DS # ) p-m(°C) (s –1 ) (kJ/mol) (kJ/mol) (kJ/mol) (kJ/mol) (kJ/mol) (kJ/mol)Tryps<strong>in</strong> psychrophile Greenland cod 5 82.0 57.7 33.0 –24.5 –2.5 –20.9 –18.3mesophile bov<strong>in</strong>e 27.7 60.2 53.9 –6.2Tryps<strong>in</strong> psychrophile Antarctic cod 15 0.97 70.5 40.6 –29.9 –0.9 –42.0 –41.2mesophile bov<strong>in</strong>e 0.68 71.4 82.6 11.3Amylase psychrophile Alteromonas 15 495.2 55.6 73.6 18.0 –4.1 –26.0 –21.9mesophile Bacillus 90.5 59.7 99.6 39.9Subtilis<strong>in</strong> sychrophile Bacillus TA41 15 25.4 62.7 36 –26.7 –3.7 –10.0 –6.3mesophile B. subtillis 5.4 66.4 46 –20.4Glucose- psychrophile Antarctic fish 0 106.5 56.1 36.8 –19.3 –2.1 –15.1 –13.06-phosphate mesophile human 42.4 58.2 51.9 –6.3dehydrogenaseXylanase psychrophile Antarctic yeast 5 14.8 61.7 45.4 –16.3 –2.6 –4.5 –1.9mesophile yeast 4.9 64.3 49.9 –14.4Chi<strong>to</strong>biase psychrophile Arthrobacter 15 98.0 59.5 44.7 –14.8 –4.0 –26.8 –22.8mesophile S. marcescens 18.0 63.5 71.5 8.0Chit<strong>in</strong>ase A psychrophile Arthrobacter 15 1.7 69.2 60.2 –9.0 2.0 –14.1 –16.1mesophile S. marcescens 3.9 67.2 74.3 7.1Glutamate psychrophile Antarctic fish 5 3.8 64.8 33.4 –31.4 –3.3 –25.6 –22.3dehydrogenase mesophile bov<strong>in</strong>e 0.9 68.1 59.0 –9.1
CMLS, Cell. Mol. Life Sci. Vol. 60, 2003 <strong>Review</strong> Article 653DH # values (table 2) and therefore reduce the temperaturedependence of k cat and the <strong>in</strong>hibi<strong>to</strong>ry effect of low temperatureson enzyme reaction rates. Thus, the decrease ofthe activation enthalpy <strong>in</strong> the enzymatic reaction of <strong>psychrophilic</strong><strong>enzymes</strong> can be considered as the ma<strong>in</strong> adaptivecharacter <strong>to</strong> low temperatures. This decrease <strong>in</strong> activationenergy is achieved structurally by a decrease <strong>in</strong> thenumber of enthalpy-driven <strong>in</strong>teractions that have <strong>to</strong> bebroken dur<strong>in</strong>g the activation steps. These <strong>in</strong>teractions alsocontribute <strong>to</strong> the stability of the folded prote<strong>in</strong> conformationand, as a corollary, the structural doma<strong>in</strong> of the enzymebear<strong>in</strong>g the active site should be less stable. This isthe first <strong>in</strong>sight suggest<strong>in</strong>g that activity and stability arel<strong>in</strong>ked <strong>in</strong> the process of thermal adaptation. As far as theactivation entropy is concerned, the negative value ofD(DS # ) p–m can also be <strong>in</strong>terpreted assum<strong>in</strong>g a more resilientactive site from <strong>psychrophilic</strong> <strong>enzymes</strong> [25]. As aconsequence of active-site flexibility, the ground state enzyme-substratecomplex ES occupies a broader distributionof conformational states translated <strong>in</strong><strong>to</strong> <strong>in</strong>creased entropyof this state compared <strong>to</strong> that of the mesophilic homologues,lead<strong>in</strong>g <strong>to</strong> a negative value of D(DS # ) p–m .Adaptive drift and adaptive optimization of K mAs <strong>in</strong>ferred from the previous section, a broader distributionof the ground state ES should be accompanied by aweaker substrate b<strong>in</strong>d<strong>in</strong>g strength if <strong>in</strong>creases <strong>in</strong> k cat arereached through active-site resilience. In other words,there is an evolutionary pressure on K m <strong>to</strong> <strong>in</strong>crease <strong>in</strong> order<strong>to</strong> maximize the overall reaction rate [73]. If the substrateis weakly bound, the ground state ES complex willfall <strong>in</strong> a less deep energy pit and, as a consequence, the activationenergy of the reaction (DG # ) will be smaller, thatis k cat will be <strong>in</strong>creased (eq. 2). This is precisely what isfound <strong>in</strong> <strong>psychrophilic</strong> <strong>enzymes</strong> which display identicalsubstrate-b<strong>in</strong>d<strong>in</strong>g site and active-site architecture (<strong>in</strong>terms of side cha<strong>in</strong> identity) when compared with theirmesophilic homologues. This fundamental notion wasfirst <strong>in</strong>troduced <strong>in</strong> the study of fish lactate dehydrogenases(A 4 -LDHs). The active-site residues are fully conserved<strong>in</strong> A 4 -LDHs from South American and Antarcticno<strong>to</strong>thenoid fish, whereas the <strong>cold</strong>-adapted <strong>enzymes</strong> arecharacterized by higher k cat and K m constants. The conclusionwas that temperature-adaptive <strong>in</strong>creases <strong>in</strong> k cat occurconcomitantly with <strong>in</strong>creases <strong>in</strong> K m <strong>in</strong> <strong>cold</strong>-activeLDHs [73]. A second and even more conclusive exampleis given by the above-mentioned structures of <strong>cold</strong>-activeand porc<strong>in</strong>e a-amylases <strong>in</strong> complex with a large <strong>in</strong>hibi<strong>to</strong>r[63, 64]. Aga<strong>in</strong>, all am<strong>in</strong>o acids form<strong>in</strong>g the active site arestrictly conserved, although the <strong>cold</strong>-active enzyme possesseshigher k cat and K m . Worth mention<strong>in</strong>g is that this aspectseems <strong>to</strong> be valid for b<strong>in</strong>d<strong>in</strong>g constants for cofac<strong>to</strong>rs,allosteric effec<strong>to</strong>rs and ions [37, 42, 44, 74].Several <strong>enzymes</strong> counteract this adaptive drift of K m <strong>to</strong>ma<strong>in</strong>ta<strong>in</strong> or <strong>to</strong> improve the substrate-b<strong>in</strong>d<strong>in</strong>g aff<strong>in</strong>ity byam<strong>in</strong>o acid substitutions with<strong>in</strong> the active site. The firstreason for these <strong>enzymes</strong> <strong>to</strong> ‘react’ aga<strong>in</strong>st the drift is obviouswhen consider<strong>in</strong>g the regula<strong>to</strong>ry function associatedwith K m , especially for <strong>in</strong>tracellular <strong>enzymes</strong>. Thesecond reason is related <strong>to</strong> the temperature dependence ofweak <strong>in</strong>teractions. Substrate b<strong>in</strong>d<strong>in</strong>g is an especially temperature-sensitivestep because both the b<strong>in</strong>d<strong>in</strong>g geometryand <strong>in</strong>teractions between b<strong>in</strong>d<strong>in</strong>g site and ligand aregoverned by weak <strong>in</strong>teractions hav<strong>in</strong>g sometimes oppositetemperature dependencies. Hydrophobic <strong>in</strong>teractionsform endothermically and are weakened by a decrease <strong>in</strong>temperature. In contrast, <strong>in</strong>teractions of an electrostaticnature (ion pairs, hydrogen bonds, Van der Waals <strong>in</strong>teractions)form exothermically and are stabilized at low temperatures.Therefore, low temperatures not only <strong>in</strong>hibitthe enzyme activity (k cat ) but can also severely alter thesubstrate-b<strong>in</strong>d<strong>in</strong>g mode and strength accord<strong>in</strong>g <strong>to</strong> thetype of <strong>in</strong>teraction <strong>in</strong>volved.The chi<strong>to</strong>biase from Antarctic bacteria nicely illustratesboth aspects as well as the extent of the k<strong>in</strong>etic optimizationthat can be reached dur<strong>in</strong>g <strong>cold</strong> adaptation of <strong>enzymes</strong>[75]. As shown <strong>in</strong> figure 2, the k cat of the <strong>cold</strong>-activechi<strong>to</strong>biase is 8 times higher than that of a mesophilicchi<strong>to</strong>biase at 5°C. However, the K m for the substrate is 25times lower at this temperature and as a result, the k cat /K mfor the <strong>cold</strong>-active enzyme is nearly 200 times greater atlow temperature. In addition, the cross-shaped plot of K mshows that the K m of each enzyme tends <strong>to</strong> m<strong>in</strong>imal andoptimal values <strong>in</strong> the range of the correspond<strong>in</strong>g environmentaltemperatures, reflect<strong>in</strong>g the f<strong>in</strong>e tun<strong>in</strong>g of this parameterreached <strong>in</strong> the course of thermal adaptation. Inthe case of the mesophilic chi<strong>to</strong>biase, the 3D structure <strong>in</strong>dicatesthat two tryp<strong>to</strong>phan residues are the ma<strong>in</strong> substrate-b<strong>in</strong>d<strong>in</strong>gligands and perform hydrophobic <strong>in</strong>teractionswith the substrate. This can be related <strong>to</strong> thedecrease of K m with temperature, accord<strong>in</strong>g <strong>to</strong> the abovementionedthermal dependence of hydrophobic <strong>in</strong>teractions.Interest<strong>in</strong>gly, the two tryp<strong>to</strong>phan residues are notfound <strong>in</strong> the <strong>cold</strong>-active chi<strong>to</strong>biase but are replaced by polarresidues that are able <strong>to</strong> perform stronger <strong>in</strong>teractionsas the temperature decreases. Such replacements may expla<strong>in</strong>the cross-shaped plot, reveal<strong>in</strong>g a subtle adaptivestrategy that takes advantage of the thermodynamic propertiesof the bonds <strong>in</strong>volved <strong>in</strong> substrate b<strong>in</strong>d<strong>in</strong>g at lowtemperatures.A work<strong>in</strong>g hypothesisThe previous sections give the impression that two ma<strong>in</strong>types of adaptation occur <strong>in</strong> <strong>psychrophilic</strong> <strong>enzymes</strong>. Onthe one hand, <strong>enzymes</strong> that possess strictly identicalresidues <strong>in</strong> the active site as their mesophilic homo-
654 G. Feller Cold-active <strong>enzymes</strong>Figure 3. Correlation of the k<strong>in</strong>etic parameters <strong>in</strong> a <strong>psychrophilic</strong>enzyme (AHA), its stabilized mutants (closed circles) and amesophilic homologue (PPA). The general trend displayed by thestabilized mutants of AHA is <strong>to</strong> decrease both k cat and K m values[65].can counteract the adaptive drift of K m <strong>to</strong> meet other physiologicalrequirements.Mutational analysis of <strong>cold</strong>-activity determ<strong>in</strong>antsFigure 2. Temperature dependence of the k<strong>in</strong>etic parameters for<strong>psychrophilic</strong> () and mesophilic () chi<strong>to</strong>biases (from Arthrobactersp. and Serratia marcescens, respectively). Data for the catalyticrate constant k cat (a), the Michaelis parameter K m (b), note the differentscales used and the relative catalytic efficiency k cat /K m (psychrophile/mesophile)(c). The <strong>cold</strong>-adapted chi<strong>to</strong>biase is characterizedby a higher activity, an optimal K m value at low temperaturesand a 200-fold higher catalytic efficiency at 7°C [75].logues, possibly because the constra<strong>in</strong>ts imposed by thestructure and the enzyme function do not allow any adjustmentsat this level. On the other hand, some <strong>enzymes</strong>possess am<strong>in</strong>o acid replacements <strong>in</strong> the active site thatcan contribute <strong>to</strong> <strong>cold</strong> activity. Accord<strong>in</strong>g <strong>to</strong> the abovementionedenergetic aspects of enzyme-catalyzed reactionsat low temperature, a work<strong>in</strong>g hypothesis can be formulated.The high catalytic activity of <strong>psychrophilic</strong> <strong>enzymes</strong>corresponds <strong>to</strong> a decrease of DG # of the catalyzedreaction (eq. 2). For all <strong>cold</strong>-active <strong>enzymes</strong>, this decreaseof DG # is achieved by a decrease of DH # (table 2)that renders the catalyzed reaction less temperature sensitive,but requires a more flexible and therefore heat-labileactive site. As a result of this active site resilience, the entropiccontribution becomes larger and substrate b<strong>in</strong>d<strong>in</strong>gis weakened for <strong>enzymes</strong> devoid of adaptive mutations <strong>in</strong>the active site. This weak b<strong>in</strong>d<strong>in</strong>g also contributes <strong>to</strong> thedecrease <strong>in</strong> DG # as the ES complex falls <strong>in</strong> a less deep energypit. However, specific mutations <strong>in</strong> the active siteAn experimental demonstration of the relationships betweenk cat and K m was recently provided us<strong>in</strong>g <strong>psychrophilic</strong>and pig pancreatic a-amylases as models [65].Stabiliz<strong>in</strong>g weak <strong>in</strong>teractions found <strong>in</strong> the porc<strong>in</strong>e a-amylase, but absent <strong>in</strong> the <strong>cold</strong>-active a-amylase, wereeng<strong>in</strong>eered by site-directed mutagenesis <strong>in</strong> the <strong>psychrophilic</strong>enzyme at positions far from the catalytic center.As shown <strong>in</strong> figure 3, stabiliz<strong>in</strong>g and rigidify<strong>in</strong>g the<strong>cold</strong>-active a-amylase tends <strong>to</strong> decrease the k cat valuesand <strong>to</strong> decrease concomitantly the K m values of the mutant<strong>enzymes</strong>, reveal<strong>in</strong>g the high correlation betweenthese two k<strong>in</strong>etic parameters. This is also another strong<strong>in</strong>dication that structural flexibility is an essential featurerelated <strong>to</strong> catalysis at low temperatures <strong>in</strong> <strong>psychrophilic</strong><strong>enzymes</strong>. One should note that other mutational studiesof <strong>cold</strong>-active <strong>enzymes</strong> have been less conclusive. Citratesynthase [76], subtilis<strong>in</strong> [44], alkal<strong>in</strong>e phosphatase [77,78], ribonuclease [79] and chit<strong>in</strong>ase (unpublished results)have been eng<strong>in</strong>eered <strong>to</strong> check the <strong>in</strong>volvement of specificadaptive mutations with<strong>in</strong> or close <strong>to</strong> the active site.These studies have revealed a complex pattern of effectson k<strong>in</strong>etic parameters, activation energy and stability thatcannot be <strong>in</strong>terpreted <strong>in</strong> simple terms. Double mutantshave also demonstrated an unsuspected synergy between<strong>in</strong>dividual mutations sometimes giv<strong>in</strong>g rise <strong>to</strong> non-additiveor opposite effects. These <strong>in</strong>terest<strong>in</strong>g studies ma<strong>in</strong>lyhighlight our current <strong>in</strong>ability <strong>to</strong> predict the effect of s<strong>in</strong>gleside cha<strong>in</strong> replacement based on rational design of the
CMLS, Cell. Mol. Life Sci. Vol. 60, 2003 <strong>Review</strong> Article 655active site, not only <strong>in</strong> <strong>psychrophilic</strong> catalysts but <strong>in</strong> <strong>enzymes</strong><strong>in</strong> general. However, a s<strong>in</strong>gle mutation <strong>in</strong> the calcium-b<strong>in</strong>d<strong>in</strong>gsite of subtilis<strong>in</strong> [44] and <strong>in</strong> the phosphateb<strong>in</strong>d<strong>in</strong>ghelix of triose phosphate isomerase [51] wereshown <strong>to</strong> <strong>in</strong>crease drastically the thermostability of the<strong>psychrophilic</strong> mutants. These results underl<strong>in</strong>e the strong<strong>in</strong>volvement of bound cofac<strong>to</strong>rs <strong>in</strong> the modulation of stabilityand activity.Table 3. Thermodynamic parameters for the irreversible <strong>in</strong>activationat 52°C of Antarctic fish and bov<strong>in</strong>e L-glutamate dehydrogenases[80].Enzyme k i E a DG # DH # TDS #(m<strong>in</strong> –1) (kJ/mol) (kJ/mol) (kJ/mol) (kJ/mol)C. aceratus 0.230 659 94.9 657 562Ox 0.043 493 99.4 490 391Heat-labile activity and weak structural stabilityThe fast <strong>in</strong>activation of <strong>cold</strong>-active <strong>enzymes</strong> by temperaturehas been recognized for decades. As a rule, the halflifeof activity for most <strong>psychrophilic</strong> <strong>enzymes</strong> at50–60°C is about 10 m<strong>in</strong>, and a few hours at 37°C. However,some <strong>cold</strong>-active <strong>enzymes</strong> already denature at20–25°C. As it counteracts the <strong>in</strong>crease of activity withtemperature, this marked heat lability is responsible forthe shift of the apparent optimal temperature of activityshown <strong>in</strong> figure 1. The irreversible denaturation betweenthe native state N and the denatured <strong>in</strong>active state I can berepresented by a simple model characterized by a first-orderrate constant k i .k iN æÆ I (4)Surpris<strong>in</strong>gly, systematic analyses of this well-recognizedproperty are scarce and generally fragmental. Such ananalysis has been performed [80] for L-glutamate dehydrogenase(GDH) from the Antarctic fish Chaenocephalusaceratus (the white-blooded icefish) and for thebov<strong>in</strong>e enzyme (table 3). As anticipated, the activation energyDG # is lower for the fish enzyme, because it undergoesa faster denaturation. Because the <strong>cold</strong>-active GDHis <strong>in</strong>activated <strong>in</strong> a narrow temperature range, the slope ofthe Arrhenius plot is steep, lead<strong>in</strong>g <strong>to</strong> very high values ofboth E a and DH # and, by difference, <strong>to</strong> a high value ofTDS # . Therefore, the activity loss is entropically driven(the low DG # value arises from the unfavorable entropycontribution). At a given temperature, the activated stateof the <strong>psychrophilic</strong> enzyme is reached through a largerentropy variation, probably reflect<strong>in</strong>g an <strong>in</strong>creased localdisorder of the active site, which is the ma<strong>in</strong> driv<strong>in</strong>g forceof heat <strong>in</strong>activation. On the other hand, the larger enthalpyvariation accompany<strong>in</strong>g this process possibly reflectsthe higher cooperativity of heat <strong>in</strong>activation <strong>in</strong> the<strong>psychrophilic</strong> enzyme, orig<strong>in</strong>at<strong>in</strong>g from the lower numberof <strong>in</strong>teractions required <strong>to</strong> disrupt the active conformation.The orig<strong>in</strong> of the heat lability is obviously found <strong>in</strong> thelow stability of the structural elements form<strong>in</strong>g the activesite (localized flexibility) or <strong>in</strong> the weak conformationalstability of <strong>cold</strong>-active <strong>enzymes</strong> (global flexibility).Global and localized stability of <strong>enzymes</strong> may haveevolved <strong>in</strong>dependently [81]. Extr<strong>in</strong>sic stabilization bysolutes of lactate dehydrogenase from warm- and <strong>cold</strong>adaptedfish <strong>in</strong>dicates that the stability of the enzymemolecules is affected <strong>to</strong> the same extent whereas the k<strong>in</strong>eticparameters of <strong>cold</strong>-active lactate dehydrogenases ismore affected by cosolvents. This effect has been attributed<strong>to</strong> a flexible active site <strong>in</strong> <strong>cold</strong>-adapted <strong>enzymes</strong> thatcan be more compacted by preferential exclusion ofsolutes. Direct evidence of differences <strong>in</strong> stability betweenthe active site and the whole structure has beenprovided by microcalorimetric studies of multidoma<strong>in</strong>prote<strong>in</strong>s, as shown <strong>in</strong> figure 4. The <strong>cold</strong>-adapted chi<strong>to</strong>biasefrom the psychrophile Arthrobacter TAD 20 has amodular architecture consist<strong>in</strong>g of an active-site doma<strong>in</strong>,a galac<strong>to</strong>se-b<strong>in</strong>d<strong>in</strong>g doma<strong>in</strong> and an immunoglobul<strong>in</strong>-likedoma<strong>in</strong>, the latter two probably be<strong>in</strong>g <strong>in</strong>volved <strong>in</strong> theb<strong>in</strong>d<strong>in</strong>g on<strong>to</strong> the chit<strong>in</strong>ous exoskele<strong>to</strong>n of crustaceans.Differential scann<strong>in</strong>g calorimetry (DSC) thermograms ofthis <strong>cold</strong>-active chi<strong>to</strong>biase display both a heat-labile anda heat-stable doma<strong>in</strong> correspond<strong>in</strong>g <strong>to</strong> the catalytic doma<strong>in</strong>and <strong>to</strong> the b<strong>in</strong>d<strong>in</strong>g doma<strong>in</strong>s, respectively, as demonstratedby saccharide-b<strong>in</strong>d<strong>in</strong>g experiments [75]. The precursorof the <strong>psychrophilic</strong> a-amylase consists of the nativeenzyme and an extra doma<strong>in</strong> <strong>in</strong>volved <strong>in</strong> the bacterialsecretion [82]. The unfold<strong>in</strong>g profiles of the precursor(fig. 4b) also display a heat-labile and a heat-stable doma<strong>in</strong>correspond<strong>in</strong>g <strong>to</strong> the native enzyme and <strong>to</strong> the secretiondoma<strong>in</strong>, respectively. These unfold<strong>in</strong>g patternsclearly demonstrate that heat lability only affects thecalorimetric doma<strong>in</strong> conta<strong>in</strong><strong>in</strong>g the active site while leav<strong>in</strong>gstability of the non-catalytic doma<strong>in</strong>s less affected orunchanged. Thus heat lability is not simply the result ofthe lack of selective pressure for stable prote<strong>in</strong> <strong>in</strong> a <strong>cold</strong>environment (as demonstrated by the stable non-catalyticdoma<strong>in</strong>s) but seems <strong>to</strong> be the consequence of the improvedplasticity required around the active site for efficientactivity at low temperatures. Another stability patterndetected <strong>in</strong> a <strong>cold</strong>-active enzyme is also worth mention<strong>in</strong>g.The <strong>psychrophilic</strong> PGK from PseudomonasTACII 18 is a globular enzyme composed of two doma<strong>in</strong>sconnected by a h<strong>in</strong>ge region that bends dur<strong>in</strong>g catalysisform<strong>in</strong>g the active site at the <strong>in</strong>terface of the two doma<strong>in</strong>s.Surpris<strong>in</strong>gly, one doma<strong>in</strong> is heat labile whereas the seconddoma<strong>in</strong> has the same stability as mesophilic PGKs(fig. 4c). The heat-labile doma<strong>in</strong> may provide the re-
656 G. Feller Cold-active <strong>enzymes</strong>Figure 5. Thermal unfold<strong>in</strong>g of a-amylases recorded by DSC.When compared with the <strong>psychrophilic</strong> AHA (from P. haloplanktis),the heat-stable <strong>enzymes</strong> PPA (from pig pancreas) and BAA(from Bacillus amyloliquefaciens) are characterized by higher T m(<strong>to</strong>p of the transition) and DH cal (area under the transition) values,by a flatten<strong>in</strong>g of the transition and by the occurrence of calorimetricdoma<strong>in</strong>s <strong>in</strong>dicated by deconvolutions <strong>in</strong> th<strong>in</strong> l<strong>in</strong>es [65].Figure 4. Three examples of <strong>psychrophilic</strong> <strong>enzymes</strong> exhibit<strong>in</strong>g localflexibility. In chi<strong>to</strong>biase (a) and the a-amylase precursor (b), theactive site is embedded <strong>in</strong> the heat-labile doma<strong>in</strong>, whereas the stabilityof the non-catalytic doma<strong>in</strong>s is unaffected. In phosphoglyceratek<strong>in</strong>ase (c), the active site is formed at the <strong>in</strong>terface of both heatlabileand heat-stable doma<strong>in</strong>s by a h<strong>in</strong>ge-bend<strong>in</strong>g mechanism[48, 75].quired flexibility around the active site and favor the reactionrate, whereas the heat-stable doma<strong>in</strong> could improvesubstrate b<strong>in</strong>d<strong>in</strong>g (as <strong>in</strong>dicated by low K m values) asa result of its rigidity [48].Conformational stability of <strong>psychrophilic</strong> <strong>enzymes</strong>As a result of their weak stability, <strong>psychrophilic</strong> <strong>enzymes</strong>are valuable model compounds for fold<strong>in</strong>g studies and areconsequently <strong>in</strong>creas<strong>in</strong>gly <strong>in</strong>vestigated by more sophisticatedtechniques. Among these, DSC offers a def<strong>in</strong>itiveadvantage because several thermodynamic parametersrelated <strong>to</strong> prote<strong>in</strong> stability are recorded directly and notextrapolated from <strong>in</strong>direct data. Figure 5 displays thecalorimetric records, or thermograms, of heat-<strong>in</strong>ducedunfold<strong>in</strong>g for <strong>psychrophilic</strong>, mesophilic and heat-stablea-amylases. Several <strong>in</strong>terest<strong>in</strong>g observations can alreadybe made from these records.1) The unfold<strong>in</strong>g of the <strong>cold</strong>-adapted enzyme occurs atlower temperatures as <strong>in</strong>dicated by the T m values whichcorrespond <strong>to</strong> the temperature of half-denaturation(for a two-state process) and are given by the <strong>to</strong>p of thetransition.2) Prote<strong>in</strong> unfold<strong>in</strong>g is an endothermic process. The areaunder the curves (fig. 5) provides the <strong>to</strong>tal amount ofheat absorbed dur<strong>in</strong>g unfold<strong>in</strong>g and is given by thecalorimetric enthalpy, DH cal . This parameter corresponds<strong>to</strong> all enthalpic contributions <strong>in</strong>volved <strong>in</strong> ma<strong>in</strong>ta<strong>in</strong><strong>in</strong>gthe compact structure and is markedly lowerfor the <strong>psychrophilic</strong> enzyme. In addition, there is aclear trend for <strong>in</strong>creas<strong>in</strong>g DH cal values <strong>in</strong> the order psychrophile< mesophile < thermophile.3) The transition for the <strong>psychrophilic</strong> enzyme is sharpand symmetric whereas other <strong>enzymes</strong> are characterizedby a flatten<strong>in</strong>g of the thermograms. This is <strong>in</strong>dicativeof a pronounced cooperativity dur<strong>in</strong>g unfold<strong>in</strong>gof the <strong>psychrophilic</strong> enzyme: its structure is stabilizedby fewer weak <strong>in</strong>teractions and disruption ofsome of these <strong>in</strong>teractions strongly <strong>in</strong>fluences the entiremolecular edifice and promotes its unfold<strong>in</strong>g.4) The <strong>psychrophilic</strong> enzyme unfolds accord<strong>in</strong>g <strong>to</strong> an allor-noneprocess, reveal<strong>in</strong>g a uniformly low stability ofits architecture. By contrast, all other homologous <strong>enzymes</strong>display two <strong>to</strong> three transitions (either observableor <strong>in</strong>dicated by deconvolution of the heat capacityfunction <strong>in</strong> fig. 5). Therefore, the conformation ofthese mesophilic and thermophilic <strong>enzymes</strong> conta<strong>in</strong>sstructural blocks or units of dist<strong>in</strong>ct stability that unfold<strong>in</strong>dependently.
CMLS, Cell. Mol. Life Sci. Vol. 60, 2003 <strong>Review</strong> Article 6575) Although not illustrated by figure 5, the unfold<strong>in</strong>g ofthe <strong>psychrophilic</strong> enzyme is freely reversible whereasall other homologous <strong>enzymes</strong> are irreversibly unfoldedafter a DSC experiment. The weak hydrophobicityof the core clusters <strong>in</strong> the <strong>cold</strong>-adapted enzymeand the low melt<strong>in</strong>g temperature, at which hydrophobic<strong>in</strong>teractions are restra<strong>in</strong>ed, certa<strong>in</strong>ly account forthis reversible character because, unlike mesophilic a-amylases, aggregation does not occur.From this microcalorimetric study, one can conclude thatthe <strong>psychrophilic</strong> enzyme <strong>in</strong>vestigated possesses a fragilemolecular edifice that is uniformly unstable and stabilizedby fewer weak <strong>in</strong>teractions than homologous mesophilic orthermophilic prote<strong>in</strong>s [65, 69]. The contribution of <strong>in</strong>dividualweak <strong>in</strong>teraction <strong>to</strong> the above-mentioned behaviorwas analyzed by site-directed mutagenesis of the <strong>psychrophilic</strong>a-amylase [65]. Fourteen mutants of this enzymewere constructed, each bear<strong>in</strong>g an eng<strong>in</strong>eeredresidue form<strong>in</strong>g a weak <strong>in</strong>teraction found <strong>in</strong> mesophilic a-amylases but absent <strong>in</strong> the <strong>cold</strong>-active a-amylase. S<strong>in</strong>gleam<strong>in</strong>o acid side cha<strong>in</strong> substitutions were found <strong>to</strong> significantlymodify the melt<strong>in</strong>g po<strong>in</strong>t T m , the calorimetric enthalpyDH cal , the cooperativity and reversibility of unfold<strong>in</strong>gbut also the thermal <strong>in</strong>activation rate constant k i and thek<strong>in</strong>etic parameters k cat and K m . Thus, a fasc<strong>in</strong>at<strong>in</strong>g aspect ofthis study was the ability <strong>to</strong> dissect the contribution of <strong>in</strong>dividualam<strong>in</strong>o acid side cha<strong>in</strong>s <strong>to</strong> the structural propertiesof heat-labile and heat-stable prote<strong>in</strong>s.Thermodynamic stability of <strong>psychrophilic</strong> <strong>enzymes</strong>The reversible unfold<strong>in</strong>g pathway of some prote<strong>in</strong>s betweenthe native state N and the unfolded state U is characterizedby an equilibrium constant K.KN ¨Æ U (4)The free energy of conformation or free energy of unfold<strong>in</strong>gis therefore:DG N–U = – RT lnK= DH N–U – TDS N–U(6)The latter relation can be rewritten for any temperature(T) us<strong>in</strong>g the parameters determ<strong>in</strong>ed experimentally byDSC:DG N–U (T) = DH cal (1–T/T m ) + DCp(T–T m )– TDCp ln (T/T m ) (7)where DCp is the difference <strong>in</strong> heat capacity between thenative and the unfolded state. This parameter reflects thehydration of non-polar groups that are exposed <strong>to</strong> waterupon unfold<strong>in</strong>g and is determ<strong>in</strong>ed by another set ofcalorimetric experiments [83].Figure 6. Gibbs free energy of unfold<strong>in</strong>g or conformational stability.Stability curves for a <strong>psychrophilic</strong> a-amylase (heavy l<strong>in</strong>e), severalmesophilic prote<strong>in</strong>s (cont<strong>in</strong>uous l<strong>in</strong>es) and a thermophilic prote<strong>in</strong>(dashed l<strong>in</strong>e). To compare prote<strong>in</strong>s of various sizes, the free energyis expressed <strong>in</strong> specific units per mole of residue [69].The stability curves, i.e. the Gibbs free energy of unfold<strong>in</strong>gas a function of temperature, for <strong>psychrophilic</strong>,mesophilic and thermophilic prote<strong>in</strong>s are drawn <strong>in</strong> figure6. These curves correspond <strong>to</strong> the energy required <strong>to</strong> disruptthe prote<strong>in</strong> structure at any given temperature. This isa parabolic function limited by the temperature of <strong>cold</strong> denaturationT m’ (below 0°C, not shown) and by the high temperaturemelt<strong>in</strong>g po<strong>in</strong>t T m , at which by def<strong>in</strong>ition DG N–U =0 [84]. Aga<strong>in</strong>, these curves provide valuable <strong>in</strong>sights <strong>in</strong><strong>to</strong>the molecular <strong>adaptations</strong> of <strong>psychrophilic</strong> <strong>enzymes</strong>.1) In pr<strong>in</strong>ciple, the low stability of a prote<strong>in</strong> can bereached via three ways (or their comb<strong>in</strong>ation): by shift<strong>in</strong>gthe stability curve <strong>to</strong>ward low temperatures (with asimilar maximal value of DG N–U as mesophiles but atmuch lower temperatures), by <strong>in</strong>creas<strong>in</strong>g the steepnessof the stability curve (with a similar maximal value ofDG N–U as mesophiles, at the same temperature but witha lower T m ) or by a global collapse of the stabilitycurve. Each of these alternatives corresponds <strong>to</strong> acomb<strong>in</strong>ation of alterations <strong>in</strong> the DH cal , T m or DCp values<strong>in</strong> equation 7. Obviously, the stability curve of the<strong>cold</strong>-active a-amylase follows the third alternative: thestrik<strong>in</strong>gly weak conformational energy (DG N–U max =22 cal/mol of residue or 92 J/mol of residue for 453am<strong>in</strong>o acids) over the entire temperature range wherethe native state prevails, i.e. between both melt<strong>in</strong>gpo<strong>in</strong>ts, arises from low values of both DH cal and T m ,whereas DCp is not significantly affected.2) The temperature for maximal prote<strong>in</strong> stability does notusually correspond <strong>to</strong> the environmental temperatureof psychrophiles, mesophiles or thermophiles but is,rather, close <strong>to</strong> room temperature [7]. The temperatureexperienced by mesophiles and thermophiles lies onthe right side of the bell-shaped stability curve, pro-
658 G. Feller Cold-active <strong>enzymes</strong>vid<strong>in</strong>g evidence that their <strong>enzymes</strong> reach the necessarybalance between optimal stability (lead<strong>in</strong>g <strong>to</strong> a compactmolecule) and flexibility (required for the catalyticfunction) by us<strong>in</strong>g the thermal dissipative force,responsible for unfold<strong>in</strong>g at higher temperatures. Incontrast, the environmental temperatures, around 0°C,that are encountered by psychrophiles are found far onthe left side of the stability curve. Mention needs <strong>to</strong> bemade at this po<strong>in</strong>t that the decrease <strong>in</strong> stability with<strong>in</strong>this region of the curve shown <strong>in</strong> figure 6, and ultimatelylead<strong>in</strong>g <strong>to</strong> <strong>cold</strong> denaturation, arises from thehydration of prote<strong>in</strong> groups which destabilizes the prote<strong>in</strong>at decreas<strong>in</strong>g temperature [85]. It follows that theorig<strong>in</strong> of flexibility of <strong>psychrophilic</strong> <strong>enzymes</strong> at lowtemperatures <strong>in</strong>volves ma<strong>in</strong>ly group hydration, and istherefore drastically different from mesophilic andthermophilic prote<strong>in</strong>s, the latter tak<strong>in</strong>g advantage ofthe conformational entropy rise with temperature <strong>to</strong>ga<strong>in</strong> mobility. The improved <strong>in</strong>teractions with the solventsuggested by the crystal structure of some <strong>cold</strong>active<strong>enzymes</strong> re<strong>in</strong>force the idea that group hydrationplays an essential role <strong>in</strong> the acquisition of flexibility<strong>in</strong> <strong>psychrophilic</strong> prote<strong>in</strong>s.3) A surpris<strong>in</strong>g consequence of the free energy functionfor the <strong>psychrophilic</strong> a-amylase shown <strong>in</strong> figure 6 isits weak stability at low temperatures when comparedwith mesophilic and thermophilic prote<strong>in</strong>s, whereas<strong>in</strong>tuitively, <strong>cold</strong>-active prote<strong>in</strong>s were also expected <strong>to</strong>be <strong>cold</strong> stable. This prote<strong>in</strong> is <strong>in</strong> fact both heat and <strong>cold</strong>labile. Assum<strong>in</strong>g constant properties of the solvent below0°C (i.e. no freez<strong>in</strong>g) and the absence of protectiveeffects from cellular components, this a-amylaseshould unfold at –10°C. Incidentally, this temperatureclosely corresponds <strong>to</strong> the probable lower limit of bacterialgrowth and, therefore, <strong>cold</strong> denaturation of somekey <strong>enzymes</strong> <strong>in</strong> psychrophiles is an additional, thoughunsuspected fac<strong>to</strong>r fix<strong>in</strong>g the lower limit of life at lowtemperatures.4) The <strong>psychrophilic</strong> a-amylase has reached a state close<strong>to</strong> the lowest possible stability of the native state [65,69]. This fact <strong>in</strong>dicates that the <strong>cold</strong>-active a-amylasecould not be any less stable than it is. If <strong>psychrophilic</strong><strong>enzymes</strong> have <strong>in</strong>deed ga<strong>in</strong>ed <strong>in</strong> flexibility at the expenseof stability <strong>in</strong> the course of evolution, this impliesthat the actual native state precludes further adaptation<strong>to</strong>ward a more mobile structure. This aspect accountsfor the imperfect adaptation of the catalyticfunction <strong>in</strong> <strong>psychrophilic</strong> <strong>enzymes</strong>, mentioned at thebeg<strong>in</strong>n<strong>in</strong>g of this review and illustrated <strong>in</strong> figure 1.Natural versus directed evolutionDirected evolution has contributed significantly <strong>to</strong> explorethe role of multiple and dispersed substitutions thatact synergistically <strong>to</strong> modify enzyme properties and functions[86, 87]. Basically, random mutations are <strong>in</strong>troduced<strong>in</strong> a gene and the result<strong>in</strong>g library is screened forthe acquisition or improvement of a specific property.This approach reveals mutations responsible for immediateand drastic adaptation <strong>in</strong> response <strong>to</strong> a strong selectionimposed <strong>in</strong> the labora<strong>to</strong>ry and selected by a physicochemicalproperty. In contrast, natural evolution is a veryslow process driven by complex environmental and metabolicconstra<strong>in</strong>ts, and selected by the viability or performanceof a liv<strong>in</strong>g organism. What can we learn from theresults of labora<strong>to</strong>ry evolution and from the general traitsof <strong>psychrophilic</strong> <strong>enzymes</strong>?Labora<strong>to</strong>ry evolution has demonstrated that enzyme activityand stability are not physically l<strong>in</strong>ked <strong>in</strong> prote<strong>in</strong>s aspreviously thought, giv<strong>in</strong>g rise <strong>to</strong> the proposal that thelow stability of <strong>cold</strong>-active <strong>enzymes</strong> results from geneticdrift orig<strong>in</strong>at<strong>in</strong>g from the lack of selective pressure forstable prote<strong>in</strong>s [8]. However, mutations that simultaneously<strong>in</strong>crease activity and stability are very rare [88] andproved <strong>to</strong> be extremely complex <strong>to</strong> obta<strong>in</strong> [89]. In fact, <strong>in</strong>nature, these environmental constra<strong>in</strong>ts do not exist exceptfor thermophilic <strong>enzymes</strong> that catalyze the conversionof labile metabolic <strong>in</strong>termediates. As a matter of fact,phosphoribosyl anthranilate isomerase from Thermo<strong>to</strong>gamaritima was found <strong>to</strong> be both extremely stable and active[90]. When random mutants are only screened forhigh activity at low temperature dur<strong>in</strong>g directed evolution,the selected <strong>enzymes</strong> <strong>in</strong>variably display the canonicalproperties of <strong>psychrophilic</strong> <strong>enzymes</strong> [91–96]. Thiscan be unders<strong>to</strong>od from the above-mentioned results, asimprovement of activity at low temperatures associatedwith lost of stability appears <strong>to</strong> be the most frequent andaccessible strategy at the level of the prote<strong>in</strong> molecule.An elegant demonstration of this aspect was given us<strong>in</strong>gornith<strong>in</strong>e carbamoyltransferase from Pyrococcus furiosusbecause random mutagenesis was associated with a functionalscreen<strong>in</strong>g procedure <strong>in</strong>volv<strong>in</strong>g the survival of thehost stra<strong>in</strong> [94]. Consequently, the low stability of <strong>cold</strong>active<strong>enzymes</strong> may be the simplest way <strong>to</strong> acquire activity<strong>in</strong> the absence of selection for stable prote<strong>in</strong>s. Thus,the occurrence of heat-stable <strong>psychrophilic</strong> <strong>enzymes</strong>should not be ruled out.ConclusionsThe most widely accepted hypothesis account<strong>in</strong>g for thedom<strong>in</strong>ant adaptive traits of <strong>cold</strong>-active <strong>enzymes</strong>, i.e. thehigh activity and the weak stability, suggests that there is acorrelation between the activity, the flexibility and the stabilityof the enzyme molecule. The flexible structure of<strong>psychrophilic</strong> <strong>enzymes</strong> can provide enhanced ability <strong>to</strong> undergodiscrete and fast conformational changes at low temperaturesimposed by the catalytic events. But the price <strong>to</strong>
CMLS, Cell. Mol. Life Sci. Vol. 60, 2003 <strong>Review</strong> Article 659pay for such plasticity is, of course, the low stability of thenative enzyme structure [1]. This l<strong>in</strong>k between activity,flexibility and stability, although <strong>in</strong>tuitively satisfy<strong>in</strong>g, rema<strong>in</strong>s<strong>to</strong> be proven experimentally <strong>in</strong> <strong>cold</strong>-active <strong>enzymes</strong>.Nonetheless, these relationships have found strong experimentalsupport from the comparison of thermophilic andmesophilic <strong>enzymes</strong> [97–99]. In contrasts, very few attemptshave been made <strong>to</strong> quantify and compare the expectedflexibility of <strong>cold</strong>-adapted prote<strong>in</strong>s. The rate of hydrogen/deuteriumexchange was recorded by Fourier transform<strong>in</strong>frared spectroscopy for two <strong>cold</strong>-active <strong>enzymes</strong>[32, 100] but no significant exchange rate was foundbetween <strong>psychrophilic</strong> <strong>enzymes</strong> compared with themesophilic homologues. The nature of the prote<strong>in</strong> motion<strong>in</strong>volved <strong>in</strong> temperature adaptation is complex and rema<strong>in</strong>slargely hypothetical [101]. For example, amide hydrogenexchange experiments us<strong>in</strong>g rubredox<strong>in</strong> from P. furiosus,the most thermostable prote<strong>in</strong> presently known, <strong>in</strong>dicatethat conformational fluctuations for solvent access do notdiffer from those of mesophilic homologues at the millisecondtime scale [102], therefore question<strong>in</strong>g the <strong>in</strong>volvemen<strong>to</strong>f conformational rigidity <strong>in</strong> the extreme heatstability of this prote<strong>in</strong>. Incidentally, this unexpected resultalso raises some doubts about the connection between exchangerates and conformational flexibility. Directed evolutionsuggests that low stability results from genetic driftbut, <strong>in</strong> contrast, the biophysical studies mentioned <strong>in</strong> thisreview <strong>in</strong>dicate that flexibility is an <strong>in</strong>tegral component ofthe adaptive strategy. At this stage, the emerg<strong>in</strong>g picturesuggests that <strong>psychrophilic</strong> <strong>enzymes</strong> utilize an improvedflexibility of the structures <strong>in</strong>volved <strong>in</strong> the catalytic cycle(as <strong>in</strong>dicated by the common heat-labile activity), whereasother prote<strong>in</strong> regions, if not implicated <strong>in</strong> catalysis, may ormay not be subjected <strong>to</strong> genetic drift, as nicely illustratedby the <strong>cold</strong>-active chi<strong>to</strong>biase and the a-amylase precursor(fig. 4). A better understand<strong>in</strong>g of enzyme evolution andprote<strong>in</strong> dynamics is obviously the next challenge for biochemicalstudies of extremophiles.Acknowledgements. The author acknowledges with thanks thef<strong>in</strong>ancial support of the European Union, the Région wallonne,Belgium and the Fonds National de la Recherche Scientifique,Belgium. 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