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DNA ligases are important enzymes required for cellular processes such as DNA replication, recombination, and repair. NAD(+)-dependent DNA ligases are essentially restricted to eubacteria, thus constituting an attractive target in the development of novel antibiotics. Although such a project might involve the systematic testing of a vast number of chemical compounds, it can essentially gain from the preliminary deciphering of the conformational stability and structural perturbations associated with the formation of the catalytically active adenylated enzyme. We have, therefore, investigated the adenylation-induced conformational changes in the mesophilic Escherichia coli and thermophilic Thermus scotoductus NAD(+)-DNA ligases, and the resistance of these enzymes to thermal and chemical (guanidine hydrochloride) denaturation. Our results clearly demonstrate that anchoring of the cofactor induces a conformational rearrangement within the active site of both mesophilic and thermophilic enzymes accompanied by their partial compaction. Furthermore, the adenylation of enzymes increases their resistance to thermal and chemical denaturation, establishing a thermodynamic link between cofactor binding and conformational stability enhancement. Finally, guanidine hydrochloride-induced unfolding of NAD(+)-dependent DNA ligases is shown to be a complex process that involves accumulation of at least two equilibrium intermediates, the molten globule and its precursor.

Adapting metabolic enzymes of microorganisms to low temperature environments may require a difficult compromise between velocity and affinity. We have investigated catalytic efficiency in a key metabolic enzyme (dihydrofolate reductase) of Moritella profunda sp. nov., a strictly psychrophilic bacterium with a maximal growth rate at 2°C or less. The enzyme is monomeric ( M r = 18,291), 55% identical to its Escherichia coli counterpart, and displays T m and denaturation enthalpy changes much lower than E. coli and Thermotoga maritima homologues. Its stability curve indicates a maximum stability above the temperature range of the organism, and predicts cold denaturation below 0°C. At mesophilic temperatures the apparent K m value for dihydrofolate is 50- to 80-fold higher than for E. coli , Lactobacillus casei , and T. maritima dihydrofolate reductases, whereas the apparent K m value for NADPH, though higher, remains in the same order of magnitude. At 5°C these values are not significantly modified. The enzyme is also much less sensitive than its E. coli counterpart to the inhibitors methotrexate and trimethoprim. The catalytic efficiency ( k cat /K m ) with respect to dihydrofolate is thus much lower than in the other three bacteria. The higher affinity for NADPH could have been maintained by selection since NADPH assists the release of the product tetrahydrofolate. Dihydrofolate reductase adaptation to low temperature thus appears to have entailed a pronounced trade-off between affinity and catalytic velocity. The kinetic features of this psychrophilic protein suggest that enzyme adaptation to low temperature may be constrained by natural limits to optimization of catalytic efficiency.

The gene encoding chitinase ArChiB from the Antarctic Arthrobacter sp. TAD20 has been expressed in Escherichia coli and the recombinant enzyme purified to homogeneity. In an effort to engineer cold-adapted biocatalysts through rational redesign to operate at elevated temperatures, we performed several mutations aiming to increase the rigidity of the molecular edifice of the selected psychrophilic chitinase. The mutations were designed on the basis of a homology-based three-dimensional model of the enzyme, and included an attempt to introduce a salt bridge (mutant N198K) and replacements of selected Gly residues by either Pro (mutants G93P, G254P) or Gln (G406Q). Mutant N198K resulted in a more stable protein (DeltaTm = 0.6 degrees C). Mutant G93P exhibited a DeltaTm of 1.2 degrees C, while mutants G254P and G406Q exhibited decreased stability. We conclude that the effect of mutating Gly residues on enzyme stability is rather complex and can only be understood in the context of the structural environment. Kinetic and spectroscopic analysis of these enzyme variants revealed that the kinetic parameters kcat and Km have been significantly modified.

The alpha-amylase precursor from the bacterium Pseudoalteromonas haloplanktis possesses a propeptide at the C-terminus possibly responsible for outer membrane translocation. Unlike the predicted beta-barrel of autotransporters, this C-terminal propeptide displays a noticeable alpha-helix content. It is connected to the enzyme by a disordered linker and has no significant interaction with the catalytic domain. The microcalorimetric pattern of the precursor also demonstrates that the stability of protein domains may evolve differently.

Psychrophiles, host of permanently cold habitats, display metabolic fluxes comparable to those exhibited by mesophilic organisms at moderate temperatures. These organisms have evolved by producing, among other peculiarities, cold-active enzymes that have the properties to cope with the reduction of chemical reaction rates induced by low temperatures. The emerging picture suggests that these enzymes display a high catalytic efficiency at low temperatures through an improved flexibility of the structural components involved in the catalytic cycle, whereas other protein regions, if not implicated in catalysis, may be even more rigid than their mesophilic counterparts. In return, the increased flexibility leads to a decreased stability of psychrophilic enzymes. In order to gain further advances in the analysis of the activity/flexibility/stability concept, psychrophilic, mesophilic, and thermophilic DNA ligases have been compared by three-dimensional-modeling studies, as well as regards their activity, surface hydrophobicity, structural permeability, conformational stabilities, and irreversible thermal unfolding. These data show that the cold-adapted DNA ligase is characterized by an increased activity at low and moderate temperatures, an overall destabilization of the molecular edifice, especially at the active site, and a high conformational flexibility. The opposite trend is observed in the mesophilic and thermophilic counterparts, the latter being characterized by a reduced low temperature activity, high stability and reduced flexibility. These results strongly suggest a complex relationship between activity, flexibility and stability. In addition, they also indicate that in cold-adapted enzymes, the driving force for denaturation is a large entropy change.

To elucidate the strategy of low temperature adaptation for a cold-adapted family 8 xylanase, the thermal and chemical stabilities, thermal inactivation, thermodependence of activity and conformational flexibility, as well as the thermodynamic basis of these processes, were compared with those of a thermophilic homolog. Differential scanning calorimetry, fluorescence monitoring of guanidine hydrochloride unfolding and fluorescence quenching were used, among other techniques, to show that the cold-adapted enzyme is characterized by a high activity at low temperatures, a poor stability and a high flexibility. In contrast, the thermophilic enzyme is shown to have a reduced low temperature activity, high stability and a reduced flexibility. These findings agree with the hypothesis that cold-adapted enzymes overcome the quandary imposed by low temperature environments via a global or local increase in the flexibility of their molecular edifice, with this in turn leading to a reduced stability. Analysis of the guanidine hydrochloride unfolding, as well as the thermodynamic parameters of irreversible thermal unfolding and thermal inactivation shows that the driving force for this denaturation and inactivation is a large entropy change while a low enthalpy change is implicated in the low temperature activity. A reduced number of salt-bridges are believed to be responsible for both these effects. Guanidine hydrochloride unfolding studies also indicate that both family 8 enzymes unfold via an intermediate prone to aggregation.

The enzyme ornithine carbamoyltransferase (OTCase) of Moritella abyssi (OTCaseMab), a new, strictly psychrophilic and piezophilic bacterial species, was purified. OTCaseMab displays maximal activity at rather low temperatures (23 to 25°C) compared to other cold-active enzymes and is much less thermoresistant than its homologues from Escherichia coli or thermophilic procaryotes. In vitro the enzyme is in equilibrium between a trimeric state and a dodecameric, more stable state. The melting point and denaturation enthalpy changes for the two forms are considerably lower than the corresponding values for the dodecameric Pyrococcus furiosus OTCase and for a thermolabile trimeric mutant thereof. OTCaseMab displays higher Km values for ornithine and carbamoyl phosphate than mesophilic and thermophilic OTCases and is only weakly inhibited by the bisubstrate analogue δ-N-phosphonoacetyl-L-ornithine (PALO). OTCaSeMab differs from other, nonpsychrophilic OTCases by substitutions in the most conserved motifs, which probably contribute to the comparatively high Km values and the lower sensitivity to PALO. The Km for ornithine, however, is substantially lower at low temperatures. A survey of the catalytic efficiencies (kcat/Km) of OTCases adapted to different temperatures showed that OTCaSeMab activity remains suboptimal at low temperature despite the 4.5-fold decrease in the Km value for ornithine observed when the temperature is brought from 20 to 5°C. OTCaSeMab adaptation to cold indicates a trade-off between affinity and catalytic velocity, suggesting that optimization of key metabolic enzymes at low temperatures may be constrained by natural limits., SCOPUS: ar.j, info:eu-repo/semantics/published

In the last few years, increased attention has been focused on a class of organisms called psychrophiles. These organisms, hosts of permanently cold habitats, often display metabolic fluxes more or less comparable to those exhibited by mesophilic organisms at moderate temperatures. Psychrophiles have evolved by producing, among other peculiarities, “cold-adapted” enzymes which have the properties to cope with the reduction of chemical reaction rates induced by low temperatures. Thermal compensation in these enzymes is reached, in most cases, through a high catalytic efficiency associated, however, with a low thermal stability. Thanks to recent advances provided by X-ray crystallography, structure modelling, protein engineering and biophysical studies, the adaptation strategies are beginning to be understood. The emerging picture suggests that psychrophilic enzymes are characterized by an improved flexibility of the structural components involved in the catalytic cycle, whereas other protein regions, if not implicated in catalysis, may be even more rigid than their mesophilic counterparts. Due to their attractive properties, i.e., a high specific activity and a low thermal stability, these enzymes constitute a tremendous potential for fundamental research and biotechnological applications.

The Antarctic psychrophile Pseudoalteromonas haloplanktis produces a cold-active cellulase. To date, a three-dimensional structure of a psychrophilic cellulase has been lacking. Crystallographic studies of this cold-adapted enzyme have therefore been initiated in order to contribute to the understanding of the molecular basis of the cold adaptation and the high catalytic efficiency of the enzyme at low and moderate temperatures. The catalytic core domain of the psychrophilic cellulase CelG from P. haloplanktis has been expressed, purified and crystallized and a complete diffraction data set to 1.8 A has been collected. The space group was found to be P2(1)2(1)2(1), with unit-cell parameters a = 135.1, b = 78.4, c = 44.1 A. A molecular-replacement solution, using the structure of the mesophilic counterpart Cel5A from Erwinia chrysanthemi as a search model, has been found.

Cold-Adapted Microorganisms—Definition Ecology Mechanisms of Adaptation to Cold Biotechnological Potential Keywords: cold-active enzymes; cold-adapted microorganisms; cold-induced proteins; cold-shock proteins; extreme environments; psychrophilic microorganisms; psychrotrophic microorganisms

Five psychrophilic Antarctic bacteria have been selected for their capacity to secrete exoenzymes into culture medium. These strains are able to grow from 0 to about 25° C. However, production of lipase fromMoraxella, α-amylase fromAlteromonas haloplanctis, β-lactamase fromPsychrobacter immobilis and protease fromBacillus is maximal at temperatures close to that of their environment (—2 to 4° C) and is strongly inhibited at higher temperatures. This thermal effect involves alterations in the secretory pathway in the upper range of temperatures, losses due to the enzyme thermal lability and in some cases to reduction in cell development. The apparent optimal activity temperature of these enzymes is between 30 and 40° C, i.e. about 20° C lower than that of their mesophilic counterparts.

Enzymes from psychrophilic organisms differ from their mesophilic counterparts in having a lower thermostability and a higher specific activity at low and moderate temperatures. The current consensus is that they have an increased flexibility, enhancing accommodation and transformation of the substrates at low energy costs. Here we describe the structure of the xylanase from the Antarctic bacterium Pseudoalteromonas haloplanktis at 1.3 A resolution. Xylanases are usually grouped into glycosyl hydrolase families 10 and 11, but this enzyme belongs to family 8. The fold differs from that of other known xylanases and can be described as an (alpha/alpha)(6) barrel. Various parameters that may explain the cold-adapted properties were examined and indicated that the protein has a reduced number of salt bridges and an increased exposure of hydrophobic residues. The crystal structures of a complex with xylobiose and of mutant D144N were obtained at 1.2 and 1.5 A resolution, respectively. Analysis of the various substrate binding sites shows that the +3 and -3 subsites are rearranged as compared to those of a family 8 homolog, while the xylobiose complex suggests the existence of a +4 subsite. A decreased acidity of the substrate binding cleft and an increased flexibility of aromatic residues lining the subsites may enhance the rate at which substrate is bound.

Secretion of cold-adapted α-amylase from Pseudoalteromonas haloplanktis TAB23 was studied in three Antarctic bacteria. We demonstrated that the enzyme is specifically secreted in the psychrophilic hosts even in the absence of a protein domain that has been previously reported to be necessary for α-amylase secretion in Escherichia coli . The occurrence of two different secretion pathways in different hosts is proposed.

Cold–adapted, or psychrophilic, organisms are able to thrive at low temperatures in permanently cold environments, which in fact characterize the greatest proportion of our planet. Psychrophiles include both prokaryotic and eukaryotic organisms and thus represent a significant proportion of the living world. These organisms produce cold–evolved enzymes that are partially able to cope with the reduction in chemical reaction rates induced by low temperatures. As a rule, cold–active enzymes display a high catalytic efficiency, associated however, with a low thermal stability. In most cases, the adaptation to cold is achieved through a reduction in the activation energy that possibly originates from an increased flexibility of either a selected area or of the overall protein structure. This enhanced plasticity seems in turn to be induced by the weak thermal stability of psychrophilic enzymes. The adaptation strategies are beginning to be understood thanks to recent advances in the elucidation of the molecular characteristics of cold–adapted enzymes derived from X–ray crystallography, protein engineering and biophysical methods. Psychrophilic organisms and their enzymes have, in recent years, increasingly attracted the attention of the scientific community due to their peculiar properties that render them particularly useful in investigating the possible relationship existing between stability, flexibility and specific activity and as valuable tools for biotechnological purposes.

Xylanases are generally classified into glycosyl hydrolase families 10 and 11 and are found to frequently have an inverse relationship between their pI and molecular mass values. However, we have isolated a psychrophilic xylanase that belongs to family 8 and which has both a high pI and high molecular mass. This novel xylanase, isolated from the Antarctic bacterium Pseudoalteromonas haloplanktis, is not homologous to family 10 or 11 enzymes but has 20-30% identity with family 8 members. NMR analysis shows that this enzyme hydrolyzes with inversion of anomeric configuration, in contrast to other known xylanases which are retaining. No cellulase, chitosanase or lichenase activity was detected. It appears to be functionally similar to family 11 xylanases. It hydrolyzes xylan to principally xylotriose and xylotetraose and is most active on long chain xylo-oligosaccharides. Kinetic studies indicate that it has a large substrate binding cleft, containing at least six xylose-binding subsites. Typical psychrophilic characteristics of a high catalytic activity at low temperatures and low thermal stability are observed. An evolutionary tree of family 8 enzymes revealed the presence of six distinct clusters. Indeed classification in family 8 would suggest an (alpha/alpha)(6) fold, distinct from that of other currently known xylanases.

The 46 kDa xylanase from the Antarctic microorganism Pseudoalteromonas haloplanktis is an enzyme that efficiently catalyzes reactions at low temperatures. Here, the crystallization of both the native protein and the SeMet-substituted enzyme and data collection from both crystals using synchrotron radiation are described. The native data showed that the crystals diffract to 1.3 A resolution and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 50.87, b = 90.51, c = 97.23 A. SAD data collected at the peak of the selenium absorption edge proved to be sufficient to determine the heavy-atom configuration and to obtain electron density of good quality.

Organisms living in permanently cold environments, which actually represent the greatest proportion of our planet, display at low temperatures metabolic fluxes comparable to those exhibited by mesophilic organisms at moderate temperatures. They produce cold-evolved enzymes partially able to cope with the reduction in chemical reaction rates and the increased viscosity of the medium induced by low temperatures. In most cases, the adaptation is achieved through a reduction in the activation energy, leading to a high catalytic efficiency, which possibly originates from an increased flexibility of either a selected area of or the overall protein structure. This enhanced plasticity seems in return to be responsible for the weak thermal stability of cold enzymes. These particular properties render cold enzymes particularly useful in investigating the possible relationships existing between stability, flexibility, and specific activity and make them potentially unrivaled for numerous biotechnological tasks. In most cases, however, the adaptation appears to be far from being fully achieved.

Isothermal titration calorimetry has been applied to the determination of the kinetic parameters of chitinases (EC 3.2.1.14) by monitoring the heat released during the hydrolysis of chitin glycosidic bonds. Experiments were carried out using two different macromolecular substrates: a soluble polymer of N-acetylglucosamine and the insoluble chitin from crab shells. Different experimental temperatures were used in order to compare the thermodependence of the activity of two chitinases from the psychrophile Arthrobacter sp. TAD20 and of chitinase A from the mesophile Serratia marcescens. The method allowed to determine unequivocally the catalytic rate constant k(cat), the activation energy (E-a) and the thermodynamic activation parameters (DeltaG(#), DeltaH(#), DeltaS(#)) of the chitinolytic reaction on the soluble substrate. The catalytic activity has also been determined on insoluble chitin, which displays an effect of substrate saturation by chitinases. On both substrates, the thermodependence of the activity of the psychrophilic chitinases was lower than that observed with the mesophilic counterpart. (C) 2001 Elsevier Science B.V. All rights reserved.

The gene encoding the phosphoglycerate kinase (PGK) from the Antarctic Pseudomonas sp. TACII18 has been cloned and found to be inserted between the genes encoding for glyceraldhyde-3-phosphate dehydrogenase and fructose aldolase. The His-tagged and the native recombinant PGK from the psychrophilic Pseudomonas were expressed in Escherichia coli. The wild-type and the native recombinant enzymes displayed identical properties, such as a decreased thermostability and a 2-fold higher catalytic efficiency at 25 degrees C when compared with the mesophilic PGK from yeast. These properties, which reflect typical features of cold-adapted enzymes, were strongly altered in the His-tagged recombinant PGK. The structural model of the psychrophilic PGK indicated that a key determinant of its low stability is the reduced number of salt bridges, surface charges, and aromatic interactions when compared with mesophilic and thermophilic PGK. Differential scanning calorimetry of the psychrophilic PGK revealed unusual variations in its conformational stability for the free and substrate-bound forms. In the free form, a heat-labile and a thermostable domain unfold independently. It is proposed that the heat-labile domain acts as a destabilizing domain, providing the required flexibility around the active site for catalysis at low temperatures.

Publisher Summary Temperature is one of the most important environmental factors for life. Cold-adapted or psychrophilic organisms are able to thrive at temperatures around 0°C. They are either prokaryotic or eukaryotic and represent a significant portion of the living world because temperatures over a considerable portion of our planet (example, polar and alpine regions, deep-sea waters) are below 5°C. Evolution has allowed these adapted organisms, named psychrophiles, to survive and grow in the restrictive conditions of these cold habitats. In these environments, psychrophiles display metabolic fluxes more or less comparable with those exhibited by mesophiles at moderate temperatures. The challenge for them is to counteract the reduction in chemical reaction rates due to low temperatures. This chapter describes the principles of enzyme cold-adaptation. It is well known that rates of chemical reactions decrease with temperature; this is described by the Arrhenius equation. The Arrhenius law also applies to psychrophiles but they have developed adaptation mechanisms enabling them to display, despite the low temperature of their environment, appropriate reaction rates contrary to non-adapted organisms, which would not survive or grow satisfactorily in such conditions of environmental stress.

Basic theoretical and practical aspects of activation parameters are briefly reviewed in the context of cold-adaptation. In order to reduce the error impact inherent to the transition state theory on the absolute values of the free energy (DeltaG(#)), enthalpy (DeltaH(#)) and entropy (DeltaS(#)) of activation, it is proposed to compare the variation of these parameters between psychrophilic and mesophilic enzymes, namely Delta(DeltaG(#))(p-m), Delta(DeltaH(#))(p-m) and Delta(DeltaS(#))(p-m). Calculation of these parameters from the available literature shows that the main adaptation of psychrophilic enzymes lies in a significant decrease of DeltaH(#), therefore leading to a higher k(cat), especially at low temperatures. Moreover, in all cases including cold-blooded animals, DeltaS(#) exerts an opposite and negative effect on the gain in k(cat). It is argued that the magnitude of this counter-effect of DeltaS(#) can be reduced by keeping some stable domains, while increasing the flexibility of the structures required to improve catalysis at low temperature, as demonstrated in several cold-active enzymes. This enthalpic-entropic balance provides a new approach explaining the two types of conformational stability detected by recent microcalorimetric experiments on psychrophilic enzymes.

The alpha-amylase sequences contained in databanks were screened for the presence of amino acid residues Arg195, Asn298 and Arg/Lys337 forming the chloride-binding site of several specialized alpha-amylases allosterically activated by this anion. This search provides 38 alpha-amylases potentially binding a chloride ion. All belong to animals, including mammals, birds, insects, acari, nematodes, molluscs, crustaceans and are also found in three extremophilic Gram-negative bacteria. An evolutionary distance tree based on complete amino acid sequences was constructed, revealing four distinct clusters of species. On the basis of multiple sequence alignment and homology modeling, invariable structural elements were defined, corresponding to the active site, the substrate binding site, the accessory binding sites, the Ca(2+) and Cl(-) binding sites, a protease-like catalytic triad and disulfide bonds. The sequence variations within functional elements allowed engineering strategies to be proposed, aimed at identifying and modifying the specificity, activity and stability of chloride-dependent alpha-amylases.

Chemical reaction rates often show a strong temperature dependency and a decrease of 10°C from room temperature typically divides the rate by a factor oscillating between 1.5 and 4. The decrease of the rate constant k indeed obeys an equation proposed by Svante Arrhenius as early as in 1889: $$K = A{e^{ - Ea/RT}}$$ (1) in which E a is the activation energy, R the gas constant (8.31 kJ mol−1) and T the temperature in Kelvin.

Alteromonas haloplanctis is a bacterium that flourishes in Antarctic sea-water and it is considered as an extreme psychrophile. We have determined the crystal structures of the alpha-amylase (AHA) secreted by this bacterium, in its native state to 2.0 angstroms resolution as well as in complex with Tris to 1.85 angstroms resolution. The structure of AHA, which is the first experimentally determined three-dimensional structure of a psychrophilic enzyme, resembles those of other known alpha-amylases of various origins with a surprisingly greatest similarity to mammalian alpha-amylases. AHA contains a chloride ion which activates the hydrolytic cleavage of substrate alpha-1,4-glycosidic bonds. The chloride binding site is situated approximately 5 angstroms from the active site which is characterized by a triad of acid residues (Asp 174, Glu 200, Asp 264). These are all involved in firm binding of the Tris moiety. A reaction mechanism for substrate hydrolysis is proposed on the basis of the Tris inhibitor binding and the chloride activation. A trio of residues (Ser 303, His 337, Glu 19) having a striking spatial resemblance with serine-protease like catalytic triads was found approximately 22 angstroms from the active site. We found that this triad is equally present in other chloride dependent alpha-amylases, and suggest that it could be responsible for autoproteolytic events observed in solution for this cold adapted alpha-amylase.

α-Amylase from the antarctic psychrophile Alteromonas haloplanktis is synthesized at 0 ± 2°C by the wild strain. This heat-labile α-amylase folds correctly when overexpressed in Escherichia coli , providing the culture temperature is sufficiently low to avoid irreversible denaturation. In the described expression system, a compromise between enzyme stability and E. coli growth rate is reached at 18°C.

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 0-30 degrees C 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 alpha-amylase, beta-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.

Chloride is the allosteric effector of vertebrate pancreatic and salivary alpha-amylases and of the bacterial alpha-amylase from Alteromonas haloplanctis. Activation experiments of A. haloplanctis alpha-amylase by several monovalent anions show that a negative charge, not restricted to that of Cl-, is essential for the amylolytic reaction. Engineering of the chloride binding site reveals that a basic residue is an essential component of the site. The mutation K337R alters the Cl--binding properties, whereas the mutation K337Q produces an active, chloride-independent enzyme. Comparison of the Kd values for Cl- in three homologous alpha-amylases also indicates that the binding affinity is dependent on the chloride coordination mode by this basic residue. Analysis of substrate and chloride binding according to the allosteric kinetic model shows that the chloride effector is not involved in substrate binding. By contrast, the pH dependence of activity and experiments of chemical modifications and Ca2+ inhibition show that the chloride ion is responsible for the pKa shift of catalytic groups and interacts with active site carboxyl groups.

The gene of subtilisin S41, an alkaline protease secreted by the psychrophile Bacillus TA41, encodes for a preproenzyme of 419 amino acids residues. The nucleotide sequence and NH2- and COOH-terminal amino acid sequencing of the purified enzyme indicate that the mature subtilisin S41 is composed of 309 residues with a predicted M(r) = 31,224. Subtilisin S41 shares most of its properties with mesophilic subtilisins (structure of the precursor, 52% amino acid sequence identity, alkaline pH optimum, broad specificity, Ca2+ binding) but is characterized by a higher specific activity on macromolecular substrate, by a shift of the optimum of activity toward low temperatures, and by a low thermal stability. The enzyme also differs by an acidic pI (5.3) and the presence of one disulfide bond. It is proposed that the psychrophilic enzyme possesses a more flexible molecular structure when compared to mesophilic and thermophilic subtilases in order to compensate for the reduction of reaction rates at low temperatures. The model of subtilisin S41 indeed reveals several features able to induce a more flexible, heat-labile conformation: the occurrence of four extended surface loops, a very hydrophilic surface through 11 extra Asp residues, and the lack of several salt bridges and aromatic-aromatic interactions. The low affinity of the Ca1 calcium binding site (Kd(app) = 10(-6) M), resulting possibly from one chelating side chain substitution and the stacking of Gly residues, also reflect a less compact conformation. The difference of free energy of stabilization between subtilisin S41 and a mesophilic subtilisin suggests that the balance of exo- and endothermically formed weak bonds is critical for the enzyme flexibility.

The alpha-amylase secreted by the antarctic bacterium Alteromonas haloplanctis displays 66% amino acid sequence similarity with porcine pancreatic alpha-amylase. The psychrophilic alpha-amylase is however characterized by a sevenfold higher kcat and kcat/Km values at 4 degrees C and a lower conformational stability estimated as 10 kJ.mol-1 with respect to the porcine enzyme. It is proposed that both properties arise from an increase in molecular flexibility required to compensate for the reduction of reaction rates at low temperatures. This is supported by the fast denaturation rates induced by temperature, urea or guanidinium chloride and by the shift towards low temperatures of the apparent optimal temperature of activity. When compared with the known three-dimensional structure of porcine pancreatic alpha-amylase, homology modelling of the psychrophilic alpha-amylase reveals several features which may be assumed to be responsible for a more flexible, heat-labile conformation: the lack of several surface salt bridges in the (beta/alpha)8 domain, the reduction of the number of weakly polar interactions involving an aromatic side chain, a lower hydrophobicity associated with the increased flexibility index of amino acids forming the hydrophobic clusters and by substitutions of proline for alanine residues in loops connecting secondary structures. The weaker affinity of the enzyme for Ca2+ (Kd = 44 nM) and for Cl- (Kd = 1.2 mM at 4 degrees C) can result from single amino acid substitutions in the Ca(2+)-binding and Cl(-)-binding sites and can also affect the compactness of alpha-amylase.The alpha-amylase secreted by the antarctic bacterium Alteromonas haloplanctis displays 66% amino acid sequence similarity with porcine pancreatic alpha-amylase. The psychrophilic alpha-amylase is however characterized by a sevenfold higher kcat and kcat/Km values at 4 degrees C and a lower conformational stability estimated as 10 kJ.mol-1 with respect to the porcine enzyme. It is proposed that both properties arise from an increase in molecular flexibility required to compensate for the reduction of reaction rates at low temperatures. This is supported by the fast denaturation rates induced by temperature, urea or guanidinium chloride and by the shift towards low temperatures of the apparent optimal temperature of activity. When compared with the known three-dimensional structure of porcine pancreatic alpha-amylase, homology modelling of the psychrophilic alpha-amylase reveals several features which may be assumed to be responsible for a more flexible, heat-labile conformation: the lack of several surface salt bridges in the (beta/alpha)8 domain, the reduction of the number of weakly polar interactions involving an aromatic side chain, a lower hydrophobicity associated with the increased flexibility index of amino acids forming the hydrophobic clusters and by substitutions of proline for alanine residues in loops connecting secondary structures. The weaker affinity of the enzyme for Ca2+ (Kd = 44 nM) and for Cl- (Kd = 1.2 mM at 4 degrees C) can result from single amino acid substitutions in the Ca(2+)-binding and Cl(-)-binding sites and can also affect the compactness of alpha-amylase.

Although the average temperature on earth is probably of the order of 15 °C, the environments with extreme temperature in which life can be considered possible are very common on the surface of the planet.

A lipase gene ( lip1 ) from the facultative psychrophilic strain Psychrobacter immobilis B10 has been cloned and sequenced. The deduced preprotein sequence is composed of 317 amino acids with a predicted M r of 35 288. A primary structure alignment of lipases including lip1 shows conserved elements for which a structural role is proposed in the light of recent crystallographic studies. The analysis of the psychrophilic enzyme sequence suggests characteristics in relation with the adaptation to cold.

The nucleotide sequence of the subtilisin-encoding gene from the antarctic psychrotroph, Bacillus TA41, was determined. The primary structure of the subtilisin precursor corresponds to a preproenzyme of 419 amino acids. Asp144, His181 and Ser359 are the proposed catalytic residues of the protease active site.

The nucleotide sequence of the subtilisin-encoding gene from the antarctic psychrotroph Bacillus TA39 was determined. The primary structure of the subtilisin precursor is composed of 420 amino acids giving rise to a mature enzyme of 309 amino acids. Asp-145, His-185 and Ser-361 are the proposed catalytic residues of the active site.

The alpha-amylase excreted by the antarctic bacterium Alteromonas haloplanctis was purified and the corresponding amy gene was cloned and sequenced. N- and C-terminal amino acid sequencing were used to establish the primary structure of the mature A. haloplanctis alpha-amylase which is composed of 453 amino acids with a predicted Mr of 49,340 and a pI of 5.5. Three Ca2+ ions are bound per molecule and its activity is modulated by chloride ions. Within the four consensus sequences, Asp-174, Glu-200, and Asp-264 are the proposed catalytic residues. The psychrotrophic A. haloplanctis alpha-amylase is characterized by a high amylolytic activity at low temperatures, a reduced apparent optimal temperature, and typical thermodynamic activation parameters A. haloplanctis alpha-amylase has also a low thermal stability as demonstrated by the temperature effect on both activity and secondary structure. It is suggested that structure flexibility and lower sensitivity of secondary structure to temperature variations in the low temperature range are the main structural adaptations of the psychrotrophic enzyme. The unusual stacking of small amino acids around the catalytic residues is proposed as a factor inducing active site flexibility and concomitant high activity of the enzyme at low temperatures.

Three parvalbumin isotypes were isolated from the white muscle of haemoglobin-myoglobin-free antarctic fish Channichthys rhinoceratus. Antibodies against the parvalbumin mixture were raised in rabbits and used for discovery, quantitation and isolation by affinity chromatography of parvalbumin in the cardiac muscle of three antarctic fish species: Channichthys rhinoceratus, Champsocephalus gunnari and Notothenia neglecta. The cardiac muscle of these species contains parvalbumin in concentration close to 1 mumol per kg wet weight.

The cloning and expression of genes from a psychrotrophic bacterium in a mesophilic host are described. Three lipase (Lip)-encoding genes (lip) from the antarctic psychrotroph, Moraxella TA144, were cloned by inserting Sau3AI-generated DNA fragments into the BamHI site of the pSP73 plasmid vector. To prevent heat denaturation of the gene product, the screening procedure on agar plates containing an emulsified lipid involved growing of Escherichia coli recombinant colonies at 25°C followed by incubation at 0°C. The three recombinant lipases (reLip) were cell-associated and differed by their respective specificity towards p-nitrophenyl esters of various aliphatic chain lengths. These cloned reLip conserved the main character of the wild-type enzymes i.e., a dramatic shift of the optimal temperature of activity towards low temperatures and pronounced heat lability.

The lip2 gene from the antarctic psychrotroph Moraxella TA144 was sequenced. The primary structure of the Lip2 preprotein deduced from the nucleotide sequence is composed of 433 amino acids with a predicted Mr of 47,222. This enzyme contains a Ser-centered consensus sequence and a conserved His-Gly dipeptide found in most lipase amino-terminal domains. These sequences are involved in the lipase active site conformation since substitution of the conserved Ser or His residues by Ala and Gln, respectively, results in the loss of both lipase and esterase activities. Structural factors that would allow proper enzyme flexibility at low temperatures are discussed. It is suggested that only subtle changes in the primary structure of these psychrotrophic enzymes can account for their ability to catalyze lipolysis at temperatures close to 0°C.

A lipase gene (lip3) from the psychotrophic strain Moraxella TA144 has been cloned and sequenced. The deduced primary structure of the lipase preprotein is composed of 315 amino acids with a predicted Mr of 34 772. This enzyme contains two consensus peptides showing cluster of glycine residues that may be involved in domain flexibility. The cloned gene product conserves the low temperature activity and the thermolability properties of the wild enzyme.

A cold-active alpha-amylase was purified from culture supernatants of the antarctic psychrophile Alteromonas haloplanctis A23 grown at 4 degrees C. In order to contribute to the understanding of the molecular basis of cold adaptations, crystallographic studies of this cold-adapted enzyme have been initiated because a three-dimensional structure of a mesophilic counterpart, pig pancreatic alpha-amylase, already exists. alpha-Amylase from A. haloplanctis, which shares 53% sequence identity with pig pancreatic alpha-amylase, has been crystallized and data to 1.85 A have been collected. The space group is found to be C222(1) with a = 71.40 A, b = 138.88 A, and c = 115.66 A. Until now, a three-dimensional structure of a psychrophilic enzyme is lacking.

Maximum activities of energy metabolism related enzymes, myofibrillar ATPase and concentrations of carnitine, lipids and myoglobin have been assayed in heart and white muscle of the ice-fish Channichthys rhinoceratus. Results are compared with those of the redblooded Paranotothenia magellanica. Increased activities of glycolytic enzymes and extremely high lactate dehydrogenase activity have been found in icefish heart, suggesting a substantial involvement of anaerobic glycolysis under conditions of oxygen depletion. During normoxia, ATP generation is achieved via oxidative pathways of carbohydrate or fatty acid catabolism. Possible implication of the myocardium in homeostatic regulation by lactate oxidation is also discussed.

Heart mitochondria of the hemoglobin and myoglobin-free antarctic icefish are specifically noncoupled with respect to succinate oxidation but not during NADH-dependent substrate oxidation. This observation suggests the coexistence of a noncoupled respiratory chain specialized in succinate oxidation and of the energy-conserving respiratory pathway. The alternative pathway of succinate would lead to the dissipation of the redox energy as heat and this thermogenic process could favour recovery after hypoxic stress periods, during which O 2 supply is severely impaired by the lack of both heme pigments.

1. 1. Trypsins from pyloric caeca of Paranotothenia magellanica and trout (Salmo gairdneri) were purified by a procedure involving acetone and ammonium sulfate fractionation followed by affinity chromatography on p-aminobenzamidine-CH-Sepharose 6B. 2. 2. Paranotothenia magellanica and trout trypsins are more stable at alkaline pH than at acid pH and they have a maximum activity around pH 8–9. Both trypsins are inhibited by classical trypsin inhibitors such as hen ovomucoid, soybean trypsin inhibitor (SBTI) and benzamidine. Trypsin from Paranotothenia magellanica is however insensitive to phenylmethylsulfonyl fluoride (PMSF). 3. 3. Paranotothenia magellanica and trout trypsins have a similar amino acid composition but their peptide maps are different. 4. 4. Over the whole range of temperatures investigated (5–35°C), Paranotothenia magellanica trypsin shows a much higher turnover number, physiological efficiency along with a lower ΔG ∗ value than those of trout and bovine trypsins.

1. 1. The troponin C (TN-C) from the Antarctic icefish (Champsocephalus gunnari) has been isolated to homogeneity by a procedure involving extraction from acetone powder, DEAE-Sepharose 4B and AcA 54 column chromatography. 2. 2. The calcium-induced conformational changes of apo TN-C have been studied by absorption difference spectroscopy, circular dichroism and intrinsic fluorescence. 3. 3. The results indicate that the overall characteristics of icefish TN-C (such as amino acid composition, modifications of helix content and of the microenvironment of aromatic residues as a function of calcium binding) are quite similar to those of rabbit TN-C. 4. 4. The intrinsic fluorescence properties are close to those reported for pike and carp TN-C.

The relative size and the ultrastructure of the heart ventricle of a haemoglobinless and myoglobinless teleost, Channichthys rhinoceratus, has been investigated. It is two to three times larger than that of a red-blooded species which also lives in the Kerguelen archipelago, Notothenia magellanica. At first sight, electron micrographs of the myocardial cell do not reveal peculiar features related to the absence of respiratory pigments. A mitochondrial compartment amounting to 18% volume fractions indicates a fairly high aerobic metabolism.

In the last few years, an increased attention has been focused on NAD+-dependent DNA ligases. This is mostly due to their potential use as antibiotic targets, because effective inhibition of these essential enzymes would result in the death of the bacterium. However, development of an efficient drug requires that the conformational modifications involved in the catalysis of NAD+-dependent DNA ligases are understood. From this perspective, we have investigated the conformational changes occurring in the thermophilic Thermus scotoductus NAD+-DNA ligase upon adenylation, as well as the effect of cofactor binding on protein resistance to thermal and chemical (guanidine hydrochloride) denaturation. Our results indicate that cofactor binding induces conformational rearrangement within the active site and promotes a compaction of the enzyme. These data support an induced “open-closure” process upon adenylation, leading to the formation of the catalytically active enzyme that is able to bind DNA. These conformational changes are likely to be associated with the protein function, preventing the formation of nonproductive complexes between deadenylated ligases and DNA. In addition, enzyme adenylation significantly increases resistance of the protein to thermal denaturation and GdmCl-induced unfolding, establishing a thermodynamic link between ligand binding and increased conformational stability. Finally, chemical unfolding of deadenylated and adenylated enzyme is accompanied by accumulation of at least two equilibrium intermediates, the molten globule and premolten globule states. Maximal populations of these intermediates are shifted toward higher GdmCl concentrations in the case of the adenylated ligase. These data provide further insights into the properties of partially folded intermediates.

Background: Enzymes from psychrophilic (cold-adapted) microorganisms operate at temperatures close to 0°C, where the activity of their mesophilic and thermophilic counterparts is drastically reduced. It has generally been assumed that thermophily is associated with rigid proteins, whereas psychrophilic enzymes have a tendency to be more flexible. Results: Insights into the cold adaptation of proteins are gained on the basis of a psychrophilic protein's molecular structure. To this end, we have determined the structure of the recombinant form of a psychrophilic α -amylase from Alteromonas haloplanctis at 2.4 a resolution. We have compared this with the structure of the wild-type enzyme, recently solved at 2.0 a resolution, and with available structures of their mesophilic counterparts. These comparative studies have enabled us to identify possible determinants of cold adaptation. Conclusions: We propose that an increased resilience of the molecular surface and a less rigid protein core, with less interdomain interactions, are determining factors of the conformational flexibility that allows efficient enzyme catalysis in cold environments.