Ž . Biochimica et Biophysica Acta 1342 1997 119–131 Review Psychrophilic enzymes: a thermodynamic challenge Charles Gerday ) , Mohamed Aittaleb, Jean Louis Arpigny, Etienne Baise, Jean-Pierre Chessa, Genevieve Garsoux, Ioan Petrescu, Georges Feller ` Laboratory of Biochemistry, Institute of Chemistry, B6 UniÕersity of Liege, Sart-Tilman, B-4000 Liege, Belgium ` ` Received 3 April 1997; revised 5 June 1997; accepted 6 June 1997 Abstract Psychrophilic microorganisms, hosts of permanently cold habitats, produce enzymes which are adapted to work at low temperatures. When compared to their mesophilic counterparts, these enzymes display a higher catalytic efficiency over a temperature range of roughly 0–308C and a high thermosensitivity. The molecular characteristics of cold enzymes originating from Antarctic bacteria have been approached through protein modelling and X-ray crystallography. The deduced three-dimensional structures of cold a-amylase, b-lactamase, lipase and subtilisin have been compared to their mesophilic homologs. It appears that the molecular adaptation resides in a weakening of the intramolecular interactions, and in some cases in an increase of the interaction with the solvent, leading to more flexible molecular edifices capable of performing catalysis at a lower energy cost. q 1997 Elsevier Science B.V. Keywords: Psychrophile; Adaptation to cold; Antarctic microorganism Contents 1. Introduction ................................................... 120 2. Psychrophilic enzyme: a definition ..................................... 121 3. Enzyme flexibility, stability and specific activity ............................. 121 4. Case studies ................................................... 125 Ž . 4.1. a-Amylase E.C. 3.2.1.1 ........................................ 125 Ž . 4.2. b-Lactamase E.C. 3.5.2.6 ....................................... 126 Ž . 4.3. Lipase E.C. 3.1.1.3 .......................................... 127 Ž . 4.4. Subtilisin E.C. 3.4.31.14 ....................................... 128 5. Conclusions ................................................... 129 ) Corresponding author. Fax: q32 4 3663364; E-mail: [email protected]0167-4838r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. Ž . PII S0167-4838 97 00093-9
Transcript
Ž .Biochimica et Biophysica Acta 1342 1997 119–131
Review
Psychrophilic enzymes: a thermodynamic challenge
Charles Gerday ), Mohamed Aittaleb, Jean Louis Arpigny, Etienne Baise, Jean-Pierre Chessa,Genevieve Garsoux, Ioan Petrescu, Georges Feller`
Laboratory of Biochemistry, Institute of Chemistry, B6 UniÕersity of Liege, Sart-Tilman, B-4000 Liege, Belgium` `
Received 3 April 1997; revised 5 June 1997; accepted 6 June 1997
Abstract
Psychrophilic microorganisms, hosts of permanently cold habitats, produce enzymes which are adapted to work at lowtemperatures. When compared to their mesophilic counterparts, these enzymes display a higher catalytic efficiency over atemperature range of roughly 0–308C and a high thermosensitivity. The molecular characteristics of cold enzymesoriginating from Antarctic bacteria have been approached through protein modelling and X-ray crystallography. Thededuced three-dimensional structures of cold a-amylase, b-lactamase, lipase and subtilisin have been compared to theirmesophilic homologs. It appears that the molecular adaptation resides in a weakening of the intramolecular interactions, andin some cases in an increase of the interaction with the solvent, leading to more flexible molecular edifices capable ofperforming catalysis at a lower energy cost. q 1997 Elsevier Science B.V.
Keywords: Psychrophile; Adaptation to cold; Antarctic microorganism
Extremophiles are mainly microorganisms experi-menting unusual environmental conditions when onetakes as references organisms living roughly underatmospheric pressure, using O as a source of elec-2
tron acceptor and metabolizing substrates at pH val-ues close to neutrality at temperatures close to theaverage temperature on earth which is about 158C.The earth’s surface is, however, dominated by lowtemperature environments, made up of extremely coldparts such as the Arctic and the Antarctic, moderatelycold parts such as mountain regions and a huge, coldand stable ecosystem, namely the marine waters whichcover 70% of the earth’s surface and display, below1000 m, temperatures not exceeding 58C indepen-dently of the latitude. All these environments arepermanently cold habitats exerting on ectothermicpopulations a highly selective pressure. They havebeen colonized, despite their extreme character, bylargely diversified organisms which have developedadaptation strategies enabling them either to surviveor to be extremely successful, like true psychrophilesŽ .from the Greek ‘psychro’scold .
An efficient adaptation, enabling metabolic fluxescomparable to those displayed by homologous organ-isms living at moderate temperatures, obviously re-quires adequate chemical reaction rates. As most ofthe chemical reactions are catalysed by enzymes, itturns out that the enzymes from psychrophilic mi-croorganisms are also adapted to cold. This meansthat their molecular structure is such that they effi-ciently catalyse chemical reactions at low tempera-ture.
One of the fields of investigation therefore coversthe understanding of the molecular adjustments whichare necessary to achieve the adaptation of these en-zymes to low temperatures. There are, however, otheraspects which are also key determinants to the adap-tation to cold. The first one is certainly membranefluidity, which has to be appropriate for the exchangeof solutes between the cell and the outer medium.
This implies a tailoring of the acyl chains of themembrane lipids corresponding to their shorteningand to an increase in their degree of saturation butalso to other complex modifications such as alteredmethyl branching and changes in the isomeric distri-bution. The problem has already been discussed by
w xRussell 1 .The third point of interest concerns the expression
of cold-shock and cold-acclimatization proteins whichhas been observed in response to either permanent,large or mild cold-shocks. Psychrophilic microorgan-isms apparently contain specific cold-shock proteinsŽ .csp completing the assortment characterizingmesophilic organisms and enabling them to harmo-niously grow in permanently cold habitats. They areobviously involved in the regulation of the activationof genes exposed to low temperatures but also toxenobiotic factors such as antibiotics. Several cspgenes like cspA, cspB and cspG have been recently
w xdescribed in E. coli 2,3 and in other microorgan-isms. Cold-shock and cold-acclimatization proteinsdefined as proteins overexpressed after all cold shocksare present in psychrotrophic bacteria such as Bacil-
w xlus cereus and Arthrobacter globiformis 4,5 ; theiressential role is still unsettled.
Next to this, recent investigations have demon-strated that some psychrophilic microorganisms likeRhodococcus erythropolis, Micrococcus cryophilusand Pseudomonas putida produce antifreeze proteinswhich probably have molecular mass close to 30kDa. A thermal hysteresis between freezing tempera-ture and growth of ice crystals was observed whenthese bacteria were grown at a temperature close to58C. The antifreeze is mainly secreted outside thecell; this presumably protects efficiently the bacterialcell against possible damages caused by the freezing
w xof the environmental water 6,7 .In this review we will lay particular emphasis on
the actual knowledge of molecular adaptations ofenzymes which are necessary to confer on psy-chrophiles the ability to be quite successful in perma-nently cold habitats.
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2. Psychrophilic enzyme: a definition
Psychrophilic and psychrotrophic microorganismshave been defined as organisms able to grow attemperatures close to 08C, but displaying differentupper growth limits around 208C for the former andaround 408C for the latter. The distinction, not reallyscientifically justified, is essentially useful to selectthe more appropriate organisms for specific studies. Itis clear that as far as molecular adaptation to cold isconcerned, the interest is to select an organism dis-playing extreme properties, meaning an upper growthlimit as low as possible. We will not make such adistinction when talking about enzymes which will beindistinctly called psychrophilic or cold enzymes be-cause their interesting properties reside in their abilityto work efficiently at low temperatures, meaningdisplaying a specific activity at low and moderatetemperatures higher than their mesophilic counter-parts. Bearing that in mind, the so-called and erro-neously called ‘optimum temperature’ has no realmeaning, being considered only as a useful relativevalue provided that the comparison between twoenzymes is carried out in strictly identical experimen-tal conditions. The apparent optimum temperature ofan enzyme indeed results from the superposition oftwo curves, one expressing the thermodependency ofactivity and the second the inactivation as a functionof temperature. This parameter has little to do withthe best growth temperature of the microorganismwhich is related to the more appropriate physiologicalconditions. This ideally should correspond to theaverage temperature of the environment. A shortdoubling time is often also erroneously related tooptimum growth conditions. Like the activity of anenzyme in vivo, they are the result of a compromisebetween the rate of enzyme-catalysed reactions oc-curring in these organisms and the stability of macro-molecular structures over a long time range.
Therefore a cold enzyme can be, and usually is,rather stable at temperatures well above the environ-mental temperature. What is important for distin-guishing a cold enzyme from its mesophilic counter-part is the higher specific activity at low temperature,probably because, in the case of psychrophilic mi-croorganisms, the selective pressure is essentiallyexerted towards the specific activity and not towardsstability factors. It is clear, however, that such an
achievement requires conformational tailoring whichcan possibly give rise to a limited stability even atmoderate temperature.
If one considers the concept that the complemen-tarity between an enzyme and its substrate is induced,a certain plasticity of the molecular edifice is alsorequired at the environmental temperature. Such aplasticity or flexibility at low temperature can only beachieved through a weakening of the intramolecularforces contributing to the cohesion of the structureleading to a relatively high instability of the enzymetowards a large variety of denaturing agents. Eachenzyme will however adopt a specific strategy so thatnumerous different situations are expected.
From the limited number of enzymes which havebeen thoroughly investigated one can say that a coldenzyme is characterized by a specific activity higherthan its mesophilic counterpart over a temperaturerange roughly covering 0–308C and by a relativeinstability.
3. Enzyme flexibility, stability and specific activity
As illustrated a long time ago by Johnston andw xWalesby 8 , looking at the specific activity and
activation parameters of Mg2q-Ca2q myofibrillarATPase from fish colonizing environments differingby their average temperature, the high specific activ-ity of cold enzymes can be explained by an activationfree energy well below that of their mesophilic coun-terparts. The low value of the activation free energyis due to a more or less severe decrease in theactivation enthalpy. The conformational changes nec-essary to reach the activation state are thereforeenergetically less costly and are dominated by adecrease in the number of enthalpy-driven interac-tions to be broken during activation. The activationentropy is, in the case of cold enzymes, either posi-tive or negative and depends not only on the 3Dstatus of the protein but also on the movement ofwater molecules making the interpretation of theentropy changes difficult. Negative modificationscould possibly correspond to an ordering effect nec-essary to render the hypothetical looser structureadequate for specific interactions with the substrates.
The higher flexibility of cold enzymes has beenprimarily deduced from the study of the thermostabil-
( )C. Gerday et al.rBiochimica et Biophysica Acta 1342 1997 119–131122
ity measured through the determination of the resid-ual activity of the enzyme exposed for some periodof time to relatively high temperatures. Usually, theloss of activity of cold enzymes is already severe at
Ž .temperatures near or below 508C Fig. 1 . One has tobe aware, however, that the loss of activity is notnecessarily strictly correlated to the unfolding of theenzyme. Only the interaction between enzyme andsubstrate could be altered without significant andirreversible modification of the three-dimensionalstructure of the enzyme. From experiments carriedout on cold a-amylase using temperature as denatur-
w xing agent 9 , the inactivation of the enzyme precedesthe modifications of conformation recorded by spec-troscopic techniques such as fluorescence emission orcircular dichroism.
To follow the three-dimensional status of the pro-tein, one has therefore to rely on appropriate tech-niques able to detect any conformational transitionsleading to inactivation. Microcalorimetry is certainlya good choice since the record of the enthalpy changesas a function of temperature reflects the changes
Ž .occurring in the folded structure Fig. 2 . Very sensi-tive differential scanning calorimeters are nowadaysavailable enabling the experiments to be carried outwith less than 100 mg of protein. Other usual andquick techniques such as fluorescence spectroscopycan also be used provided that some tryptophan
Fig. 1. Thermal stability at 508C, as measured by the residualactivity as a function or time using nitrocefin as substrate, of the
Ž .Antarctic b-lactamase from Psychrobacter immobilis A5 I
Ž .and of the mesophilic b-lactamase from E. cloacae Q908R B
in 25 mM TrisrHCl, pH 8.5, containing 100 mgrml bovineserum albumin.
Fig. 2. Heat capacity of unfolding as a function of temperature ofthe alkaline Ca2q-Zn2q protease from the Antarctic strain Pseu-domonas aeruginosa compared to that of the homologous en-zyme from the mesophile P. aeruginosa IF03455. Enzyme con-centration is 2 mgrml in 20 mM Tris, 10 mM CaCl at pH 8.2
Temperature gradient 18Crmin.
residues are in a suitable environment, the modifica-tion of which alters both the amplitude and theemission wavelength of the signal. Circular dichroismspectroscopy in the far UV region, although oftenabusively used, is not suitable since it gives anevaluation of the secondary structure and does notreflect the transitions affecting only the three-dimen-
Ž .sional structure Fig. 3 . It is however often true that,at least for single domain proteins, the various possi-ble structural forms of either the unfolded and nativestate of the enzyme are in rapid equilibrium witheach other. The unfolding of the structure is highlycooperative and can be described as an all or nonetransition. In this case, if the unfolded state is really arandom coil, CD spectroscopy in the far UV regioncan be used, to follow conformation changes in aprotein. Near UV CD spectroscopy in the aromaticregion around 270 nm can be appropriate since itprovides an evaluation of the relative freedom of thearomatic side chains. Additional problems can arisewhen multidomains proteins are concerned; in thiscase the heat absorption during denaturation can have
w xa complex profile 10 , or when the ‘unfolded’ statestill presents a more or less intact secondary structure
( )C. Gerday et al.rBiochimica et Biophysica Acta 1342 1997 119–131 123
or when stable folding intermediates displaying col-w xlapsed three-dimensional structures are observed 11 .
It is worth mentioning that a denatured protein candiffer in its optical and hydrodynamic properties de-
w xpending on the denaturing agent 12–14 . As writtenw xby Privalov 15 ‘‘Viscosity and optical properties are
not thermodynamic characteristics and do not specifythe macroscopic state; therefore, even a significantdifference in these parameters is not very meaning-ful’’.
As stated above and as already discussed byw xHochachka and Somero 16 , cold enzymes are prob-
ably characterized by an increased flexibility of thepolypeptide chain enabling an easier accommodationof substrates, especially macromolecular substrates atlow temperature. The flexibility characterizes molec-ular edifices which can adopt multiple structureshaving comparable energy so that the interconversiononly implies very small conformational changes. Toexplain the behaviour of cold enzymes, the flexibilityshould also correspond to the relative amplitude ofthe possible deformations which can occur withinsmall energy changes. Up to now, the possible higherflexibility of cold enzymes has not been demon-strated; it was only inferred from the higher ther-mosensitivity of these enzymes. Their high specific
Fig. 3. Urea-induced unfolding process of carbonic anhydrase asŽ .followed by the decrease of the negative ellipticity CD at 220
Ž .nm ` and by fluorescence spectroscopy I320rI360 or negativeŽ . Ž . Ž w x.ellipticity CD at 270 nm v adapted from Ref. 51 .
Table 1Kinetic parameters at different temperatures of some cold en-zymes from psychrophilic microorganisms compared to those ofhomologous mesophilic enzymes
k Kcat m
a y1 y1a -Amylase S g lTemperature 48C 258C 48C 258CAlteromonas haloplanctis 490 1369 1.09 1.27Ž .AntarcticPig pancreas 71 326 1.05 1.12
a Starch as substrate.b Azocasein as substrate.c Nitrocefin as substrate.d Suc-FAAF-pNA as substrate.e Oat spelt xylane as substrate.
Ž .activity Table 1 has been related to their possiblyhigher flexibility leading to their rather high instabil-ity. Support of this postulate came from the study ofenzymes from thermophilic organisms which displayhigh stability towards various denaturing agent but
w xlow specific activity at ordinary temperature 17,18 .The inverse relationship between protein stability andactivity has also been recently evaluated by site-di-rected mutagenesis carried out on T4 lysozyme; the
w xauthors concluded that this relation is general 19 .However only the residues involved in catalysis orsubstrate binding were investigated; it would be cer-tainly of interest to carry out such experiments onother areas of the protein to detect any allosteric
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effects on the stability and on the specific activity ofthe enzyme. Experiments tending first to stabilize a
w xcold subtilisin TA39 20,21 led to the conclusion thatthe single mutation transforming a poor ligand for
2q Ž . Ž .Ca T in calcium site one into a good ligand Dgave rise first to a dramatic increase of the stabilityof the cold enzyme and second, astonishingly, to afurther increase of the specific activity of the mutatedenzyme by a factor of 1.5–3 depending on the sub-strate. The introduction of new amino acids in awild-type structure often gives rise to unexpecteddata due to the cooperativity of the effects. It iscertainly possible that some parts of the moleculecontrol the stability, whereas some others are moreimplicated in the control of the activity.
Flexibility can be seen from the spread of electrondensities in crystallized structures quantified by theso-called temperature or B-factors from which were
w xderived the flexibility index F 22 . This factor is notalways related to thermostability since the latter canbe achieved through, for example, an increase in the
w xnumber of ion pairs 18 .The problem of flexibility has also been studied
through the hydrogen–deuterium exchange process;the higher the exchange rate, the higher the flexibil-ity, and the lower the stability of the enzyme. Thishas been carried out using Fourier transform infrared
Ž . w xspectroscopy FTIR 23 . High resolution NMR toevaluate 15N relaxation has also been used in the case
w xof savinase 24 . Such techniques have not yet beensystematically applied to cold enzymes. Only a fewindications have been derived from the now available
w xthree-dimensional structure of cold a-amylase 25and from FTIR spectroscopy carried out on the sameenzyme; the assumption is that a hydrogen atominvolved in an interaction with another group cannoteasily exchange.
Looking at the radiocrystallographic data, no strik-ing difference between the B-factors of cold and pigpancreatic a-amylase was observed; however thepacking is different and it is known that intermolecu-lar interactions can influence B-factors. One willhave to await further analysis of the three-dimen-sional structure to have a better evaluation of theB-factor values of key regions in the molecule.
Preliminary experiments of 1H– 2H exchange alsocarried out on the cold a-amylase from Alteromonashaloplanctis and pig pancreatic a-amylase and fol-
lowed by FTIR spectroscopy, in collaboration with E.Goormaghtig and J.M. Ruysschaert from the‘Laboratoire de Chimie physique des macromole-´cules’, Universite libre de Bruxelles, did not provide´further argument in favour of the higher flexibility ofcold enzymes. Apparently the exchange rates werefound to be similar in the cold and homeothermicenzyme giving rise to similar protection factors. Fur-ther experiments are however necessary to ascertainthis conclusion.
Another aspect of cold enzymes which has notbeen investigated so far is related to the question ofwhether or not a psychrophilic enzyme would resist
w xcold denaturation. Indeed, Privalov et al. 26 haveshown that cold denaturation is a general property ofglobular proteins. This is clearly shown in Fig. 4 inwhich the difference in Gibbs free energy betweenthe denatured and the native state D
N G is plotted as aD
function of temperature. Low pH values are used inorder to shift the temperature of cold denaturationtowards values above freezing since cold denatura-tion should take place often below the freezing pointof water. Moderate concentrations of urea have alsobeen used and quite recent investigations have shown
Ž .that a mutant of barstar C40r82A , an intracellular
Fig. 4. Gibbs energy difference as a function of temperaturebetween the denatured and the native state of metmyoglobin at
ŽpH values 3.77 and 3.95 in 10 mM sodium acetate adapted fromw x.Ref. 51 .
( )C. Gerday et al.rBiochimica et Biophysica Acta 1342 1997 119–131 125
inhibitor of barnase properly folded at 302 K, can bedenatured at 278 K in the presence of 3 M urea; theunfolded state showing, however, populated residual
w xstructures under the form of helices 27 . Such astudy has not been carried out on cold enzymes, butwould certainly be worthwhile in relation with theirpermanent exposure to low temperatures. Low tem-peratures often also give rise to dissociation of multi-
w xmeric proteins into subunits 28 . There is howeverno indication of such a phenomenon in cold enzymes
w xsuch as lactate dehydrogenase 29 or triosephosphatew xisomerase 30 . Moreover the cold-induced depolym-
erization of cytoplasmic microtubules into tubulinsobserved in mesophilic organisms is completely im-paired in microtubules from Antarctic fish. They areindeed rapidly formed from pure tubulins at tempera-tures between 0 et 208C. The adaptive changes occurat the level of the tubulin subunits. They correspondto an increased dependence of the interdimer contacts
w xon hydrophobic interactions 31 .
4. Case studies
Only cold enzymes for which enough structuralelements are available to be compared to homologouscounterparts will be discussed in this section. A fewstudies on other enzymes produced by other cold-adapted microorganisms can be found in the literaturew x29,32–34 .
( )4.1. a-Amylase E.C. 3.2.1.1
The cold a-amylase from the Antarctic psy-chrophile Alteromonas haloplanctis has been exten-
w xsively studied 35,9 . It is the first cold enzyme whichhas been successfully crystallized, the structure of
˚ w xwhich was resolved at 1.85 A resolution 25 . Theanalysis of the refined structure has not yet beenachieved so that the strategy of the adaptation to coldof this a-amylase has been deduced from a computer-ized model taking advantage of the favourable se-
Ž .quence isology 53% with pig pancreatic a-amylase.Contrary to other microbial a-amylases, the psy-chrophilic enzyme requires a Cly ion involved in theallosteric activation of the enzyme. It also requiresthe binding of one Ca2q for activity like othermesophilic enzymes and apparently contrary to hy-
w xperthermophilic a-amylases 36 .
The enzyme has a molecular mass of 49 340 Daw x35 and the polypeptide chain is a bit shorter thanthat of pig pancreatic a-amylase which has beentaken as a reference since the similarity in amino acidsequence is higher than with any other a-amylasesfrom bacterial or fungal origin. The three-dimen-sional structure is also very similar to that of pigpancreatic a-amylase showing the characteristic A, Band C domains. When comparing the structural pa-rameters which are supposed to participate in therespective stabilisation of the cold and the mam-malian enzymes, one can clearly see that the coldenzyme contains, fewer salt bridges, fewer aromaticinteractions, smaller hydrophobic clusters, fewer pro-line and arginine residues, and that the stabilisation of
w x 2qhelix dipoles is also weaker 9 . The affinity for Caand Cly is also lowered by factors close to 2000 and20, respectively. All these elements have been con-sidered to be implicated in the adaptation to cold byconferring on the cold enzyme a less cohesive struc-
w xture 9 . To corroborate this, a programme of site-di-rected mutagenesis has been carried out. The firstsites to be engineered were the Ca2q and Cly bind-ing sites. In fact, they differ only slightly from theequivalent sites of pig pancreatic a-amylase. In theCa2q coordination edifice, corresponding to a pentag-onal bipyramid, Arg-158 of pig pancreatic a-amylaseis replaced in the cold enzyme by a Gln-158. At thisposition the Ca2q is coordinated via the oxygen fromthe main chain carbonyl. In the pig pancreatic en-zyme, the lateral chain of Arg-158 is involved in asalt bridge with Glu-246; it was therefore thoughtthat the Gln in position 158 was responsible for thelow affinity of the cold enzyme for Ca2q. It wasdecided to restore in the cold enzyme an Arg inposition 158 and see what would be the effect on thestability and specific activity of the cold enzyme.Interestingly the mutation Q158R did not restore ahigh affinity for Ca2q; on the contrary, the apparentdissociation increases by a factor of 4 when com-pared to the cold enzyme. The thermostability of the
Ž .mutant is weakly lower DT s"1.58C than that ofm
the wild-type enzyme but the specific activity is alsodepressed by a factor close to 4. The lower affinity ofthe cold enzyme for Ca2q is probably not reallyimplicated in the adaptation to cold but is rather aconsequence of other changes occurring elsewhere inthe molecular edifice. It is worth mentioning that
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when the pig pancreatic a-amylase is highly stabi-lized by the binding of one Ca2q, the cation hasalmost no effect on the stability of the cold enzyme.Indeed the conformational transition followed, as afunction of temperature, by fluorescence spec-troscopy at 350 nm, indicates that in the presence ofeither 1 mM Ca2q or 5 mM EGTA, the meltingtemperature of the cold enzyme is fixed at 45"0.58C.On the contrary, for pig pancreatic a-amylase in thepresence of Ca2q the T s62"0.58C, whereas inm
the presence of EGTA the T dropped to 468C.m
Chloride is, as with other negative ions, an al-losteric effector of some a-amylases and, in theparticular case of the pig pancreatic and Antarctic A.haloplanctis a-amylases, it is essential for amylolyticactivity. The anion is located near the centre of thebra 8 barrel of domain A and is coordinated in thepig enzyme by Arg-195, Asn-298, Arg-337 and awater molecule. Arg-337 coordinates the anion in abivalent way. The coordinating site in the cold en-zyme is identical, except that Arg-337 is replaced bya Lys. The Arg–Lys replacement is part of thestrategy enabling the conversion of thermophilic intomesophilic and a fortiori psychrophilic enzymes sinceArg is able to settle as many as five weak interactionswith neighbouring groups, whereas Lys is essentially
w xmonovalent 37 . Position 337 is apparently crucialfor the binding of the halide. All chloride-dependentenzymes have indeed a basic residue at this position,either K or R, whereas Cly-independent a-amylases
Ž .display other types of residues Table 2 . From theanalysis of the three-dimensional model of cold a-amylase, it was assumed that the replacement R337Kwas at the origin of the lower affinity of the coldenzyme for Cly providing a contribution to the in-crease in flexibility of the cold enzyme. Two mutants
Ž .were produced K337R and K337Q , with the inten-tion of first restoring the coordinating site typical ofpig pancreatic a-amylase, and second mimicking sit-uations occurring in chloride-independent a-amylases. The mutant K337R still binds Cly but withan affinity reduced by a factor of 10 when compared
w xto the wild or recombinant cold enzyme 38 . Thiswas of course an unexpected result. The specificactivity of the mutated enzyme is decreased by afactor of 2 and the stability increases slightly sincethe melting temperature is higher by more or less38C. As in the case of the binding of Ca2q, the anion
Table 2Surroundings of amino acid residue 377 of several a-amylases
Ž . Ž . yinvolved basic or not other in Cl coordination
337
AHA P Y G Y P K V M S S YThecu P Y G T P K V M S S YPPA P Y G F T R V M S S YDrome P F G T P R V M S S FAnoga P Y G Q L R I M S S F
Strli P Y G S P D V H S G YBacam E S G Y P Q V F Y G DTAKA N D G L P I I Y A G QBarley H P G Y P C I F Y D H
Cly has a stabilisation effect on pig pancreatic a-amylase but none on the psychrophilic enzyme.
The mutation K337Q, as expected, completely im-y w xpairs the binding of Cl 21 , but the mutant still
displays some amylolytic activity with a k loweredcat
by a factor close to 10. In the absence of Cly, thewild-type enzyme is completely inactive but stillproperly folded as in the case of pig pancreatica-amylase. One can conclude that a basic residue inposition 337 is inhibitory unless the positive charge iscounterbalanced by a negative ion in the site. Againthe lower affinity of the cold enzyme for Cly is notreally a strategy to render the molecule more flexible,it is a consequence of other molecular alterations.
( )4.2. b-Lactamase E.C. 3.5.2.6
A b-lactamase has been recently purified from aGram-negative strain collected on organic materialabandoned for about 40 years near the fire-destroyedFrench Antarctic base Port Martin in Terre Adelie.The species was tentatively identified as Psychrobac-ter immobilis. It is worth mentioning first that theenzyme is the first b-lactamase found to be excretedin the extracellular medium by a Gram-negative strainw x39 . The enzyme has been purified, characterizedand cloned in E. coli.
Optimal growth and enzyme excretion occur bestat temperatures close to 58C. The enzyme is rapidly
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inactivated by exposure at 508C, whereas the homolo-gous enzyme from Enterococcus cloacae is stable
Ž .for hours at this temperature Fig. 1 .Ž .Using nitrocefin as substrate, one can see Table 1
that the specific activity of the cold enzyme is thehighest ever recorded for mesophilic class C b-lactamases, in agreement with the usual properties ofcold enzymes. A computerized model was built tak-ing advantage of the reasonably high percentage of
Ž .amino acid sequence identity 40% with that of E.w xcloacae b-lactamase 40 .
The folding of the two enzymes is quite similarwith an all-helical domain and a mixed arb domain.The striking differences which could be involved inthe adaptation to cold are first the lower Pro and Argcontent of the cold enzyme. Two Pro are notablymissing in the loop connecting helices a5 and a6near the active site and three other Pro, which arestrictly conserved in all mesophilic class C b-lacta-mases, are replaced by other residues. In themesophilic E. cloacae b-lactamase there are 16 argi-nine-mediated hydrogen bonds; only 10 are found inthe homologous psychrophilic enzyme. Among theweakly polar aromatic–aromatic interactions con-served in mesophilic b-lactamases and having animportant role in the stabilisation of the conforma-tion, two are missing in P.immobilis b-lactamase.Contrary also to mesophilic b-lactamases, the psy-chrophilic enzyme has an acidic isoelectric pointresulting from six extra acidic side chains which arelocated in the first 30 residues of the polypeptidechain. These residues interact with the solvent and itis known that these interactions can destabilize pro-
w xteins in general 41 . The N-terminus of the arb
domain also lacks a Pro residue. All these elementscontribute to increase the plasticity of the domainarb. A lower global hydrophobicity of the cold b
lactamase has also been deduced from the aliphaticw x w xindex 42 and the PRIFT scale 43 .
( )4.3. Lipase E.C. 3.1.1.3
Lipases produced by Antarctic cold-adapted bacte-ria have been the subject of several studies, all ofthem failing, however, to obtain purified forms due tothe difficulty of eliminating lipopolysaccharides pro-duced by the Antarctic microorganisms, and foundstrongly associated with the lipid hydrolases. The
cloning and expression of the coding genes inmesophilic organisms such as E. coli did not solvethe problem, the cold lipases apparently being ex-
w xtremely unstable within their hosts 44,45 .The investigations were, however, pursued on
semi-purified preparations of the lipase from Psy-chrobacter immobilis B10, the nucleotide sequence
w xof which is also available 46 . Some properties of thecold lipase were compared to those of the lipase fromthe mesophilic bacterium Pseudomonas aeruginosa.
The activity of the cold enzyme presents an appar-ent optimal activity around 358C and retains about20% of its activity at 08C, whereas the activity of themesophilic lipase is close to zero below 208C andstill increases at temperatures above 608C.
The activation energies evaluated from the Arrhe-nius plots are 63 and 110 kJrmol for the cold and
w xmesophilic enzymes, respectively 47 , underliningthe cold character of the lipase produced by theAntarctic bacterium. This character is also illustratedby the high thermosensitivity of the cold lipase dis-playing at 608C a half-life 2 orders of magnitudelower than that of the mesophilic enzyme. The align-ment of the amino acid sequences revealed a 21%maximum sequence identity with the dehalogenasefrom Xanthobacter autotrophicus and 11% with thatof the lipase from Pseudomonas glumae, the onlybacterial lipase with a known three-dimensionalstructure. The two reference structures fold similarly
w xat the level of domain I 48 since four helices andfive b-strands occupy identical positions in space.The second domain inserted in domain I is morevariable; it contains the helical lid of the lipase. Thecatalytic cavities are both located between the twodomains.
The cold lipase was modelled using the dehaloge-nase as a reference structure. The folding of domain Ifits very well with its equivalent in the dehalogenase.The similitude observed at the level of domain II isless spectacular due to the poor sequence similarity
Ž .with the dehalogenase Fig. 5 . The structure of thecold lipase was compared to that of the lipase fromPseudomonas glumae and to the dehalogenase fromXanthobacter autotrophicus. Parameters which couldbe involved in the adaptation to cold are the weaken-ing of the hydrophobic clusters, the dramatic decreaseŽ .40% of the Pro content and of the ratio ArgrArgqLys. Moreover when compared to the dehalogenase,
( )C. Gerday et al.rBiochimica et Biophysica Acta 1342 1997 119–131128
Fig. 5. Three-dimensional representation of the structure of theŽ .lipase from the psychrophile Psychrobacter immobilis PIL and
of the structure of the dehalogenase from the mesophile Xan-Ž .thobacter autotrophicus GJ10 XAH . One can see the striking
similarities of the respective a rb-hydrolase folds of Domain IŽ . Žlower part of the figure and of Domain II upper part of the
.figure . The His of the active site is also shown within thecatalytic cavity located between the two domains.
the cold lipase displays a very small number ofaromatic–aromatic interactions and of salt bridges.The location of some salt bridges which are absent in
the cold lipase seems to be crucial for the adaptationto cold. Firstly, two salt bridges out of four whichlink domain I and domain II of the dehalogenasedisappear in the Antarctic lipase. It is worth recallinghere that the catalytic cavity of these enzymes isprecisely located at the junction between the twodomains. Secondly, the salt bridge positioned veryclose to the Asp residue of the active site of theP.glumae lipase is also removed in the Antarcticlipase. It is concluded that the catalytic cavity of thecold lipase is characterized by a high plasticity en-abling the enzyme to accommodate easily the sub-
w xstrates at low temperatures 47 .
( )4.4. Subtilisin E.C. 3.4.31.14
The genome of the Antarctic Bacillus strain TA39contains two genes in tandem coding for two subti-lases, S39 and S41. For unknown reasons only S39 isexpressed significantly giving rise to a typical coldprotease characterized by a high thermosensitivityand a high specific activity at low temperature. Thenucleotide and derived amino acid sequences havebeen determined and a computerized model of the
w xthree-dimensional structure was built 49 . The struc-ture of S39 was compared to that of subtilisin BPNX
w xtaken as a reference 20 . The structural differenceswhich could be involved in the adaptation to cold arethe following: spectacular increase of the number of
Ž .Asp residues 21 vs. 9 at the surface of the protein,Ž .reduced number of salt bridges 5 vs. 2 , complete
lack of aromatic–aromatic interactions and muchlower affinity of site one for Ca2q. All these struc-tural modifications go in the usual direction andcould contribute to the increase in the flexibility ofthe molecular edifice. One cannot of course excludethe possibility that these alterations are related to thespecies rather than to a real adaptation to cold. This isthe reason why a programme of site-directed mutage-nesis has been started in order to check the formu-lated hypotheses. This programme is still at the be-ginning but one result already appears very signifi-cant. It concerns the substitution N™T at position185 in the cold enzyme. This residue is involved inthe Ca2q coordination through the O of the lateralchain. Threonine is a bad ligand for Ca2q and it wasthought that this substitution was responsible for thelower affinity of the cold enzyme for Ca2q.
( )C. Gerday et al.rBiochimica et Biophysica Acta 1342 1997 119–131 129
The Thr residue was therefore replaced by an Asp;a strong stabilizing effect was recorded since themutated cold enzyme displayed a thermostabilitycomparable to that of the mesophilic subtilisin. Thisin fact coincided with the increase of the affinity ofthe mutated cold enzyme for Ca2q by a factor higherthan a hundred. More surprising was the effect of theapparent rigidification of the molecular edifice on thespecific activity which in fact increases by a factor of1.5 to 3, depending on the substrate, when comparedto that of the wild type enzyme. We cannot excludethe possibility that the increase of the specific activityis due to long distance electrostatic effects inducedby the introduction of a negative charge in the cal-
w xcium binding site 50 . We can nevertheless concludethat the lower affinity of the cold enzyme for Ca2q
is, at least, partly responsible for the increased flexi-bility illustrated by the high thermosensitivity of theenzyme. Other parameters have also to be tested andin particular the surface charges. Indeed the replace-ment of the exceeding Asp residues by residues noninteracting with the solvent can also provide veryuseful information about the idea, already illustrated
w xelsewhere 41 , that protein–solvent interactions tendto destabilize the macromolecular structure. The ef-fect of the other mutations tested tending to stabilizethe structure by introducing an additional salt bridgeA181KrS210E or an additional aromatic interactionH121W did not give rise to any apparent stabilizationof the cold enzyme. Both of them however increasedthe specific activity by a factor close to 3 for yetunknown reasons. Since the stability was deducedfrom the residual activity, it is possible that theapparent lack of stabilizing effect of these mutationswas due to an increased autolysis balancing the ef-fect. One will have to use biophysical methods toevaluate the respective stability of these mutants.
5. Conclusions
The molecular adaptation to cold of enzymes origi-nating from microorganisms experiencing perma-nently cold habitats gave rise to molecular structurespresenting an increased sensitivity towards variousdenaturing agents. This is a consequence of the weak-ening of the intramolecular forces and, in certaincases, of the reinforcement of molecular interactions
with the solvent, both contributing to the instabilityof the cold enzymes.
The alteration of the molecular structure of coldenzymes is primarily designed to provide adequateplasticity of the molecule at the environmental tem-perature in order to accommodate the substrates witha minimum of energy expenditure. Indeed it is clearthat catalysis, which implies structural modificationsof the enzyme during the interaction with the sub-strate and during the transition towards the activatedstate, is efficient only if a delicate balance betweenstability and plasticity of the active site is achieved atthe considered temperature. Ideally, to obtain similarperformances at their environmental temperature, thespecific activities of enzymes produced by psy-chrophiles, mesophiles and thermophiles ought to beclose to each other at the temperature experienced bythe microorganism. We know that it is not quite thecase and, in fact, the specific activity of enzymesfrom psychrophiles is, at the environmental tempera-ture, lower than the specific activity of enzymes frommesophiles. In the case of thermophiles, the specificactivity at the ‘physiological’ temperature is higheror lower than that recorded for the homologousmesophilic enzymes contrarily to what would beexpected from the exponential thermodependency ofthe activity. This means that if a good compromisebetween activity and stability can be naturallyachieved in the case of enzymes adapted to work atmoderate temperatures, it is probably far from perfectwhen cold and hot enzymes are concerned. In thefirst case, an appropriate flexibility cannot be reachedeasily without unacceptable instability and, in thesecond case, the stiffness of the molecular structurenecessary to achieve a reasonable stability precludesin most cases the possibility to reach a high catalyticefficiency. Nature obviously has not tested all thepossibilities of compromise acting in priority towardsthe more urgent requirements, i.e. a good activity atlow temperature in the case of enzymes from psy-chrophiles and a good thermostability at high temper-ature in the case of thermophiles. Therefore stabilityon one hand and specific activity on the other handare of relatively secondary importance for psy-chrophiles and thermophiles, respectively. Researchfellows can probably perform better since they havethe technical skill to rapidly engineer enzymes fromextremophiles in such a way to associate, in the best
( )C. Gerday et al.rBiochimica et Biophysica Acta 1342 1997 119–131130
possible combination, for biotechnological purposes,stability and specific activity at any required tempera-ture.
Acknowledgements
This research has been supported by the E.U.under the form of a network contract no. ERBCHCT940521, a concerted action BIO4-CT95-0017 and aBiotech programme BIO4-CT96-0051; by the‘Ministere de l’Education, de la Recherche et de la`Formation’, concerted action ARC93r98-170 and bythe ‘Region Wallonne—Direction Generale des´ ´ ´Technologies’: Convention 1828. We also thank the‘Institut Francais de Recherche et de Technologiepolaire’ for generously accomodating our researchfellows at the French Antarctic Station J.S. Dumontd’Urville in Terre Adelie.´
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