Decreases in activation energy and substrate affinity in cold-adapted A4-lactate dehydrogenase: evidence from the Antarctic notothenioid fish Chaenocephalus aceratus

Enzyme function is strongly affected by temperature, and orthologs from species adapted to different thermal environments often show temperature compensation in kinetic properties. Antarctic notothenioid fishes live in a habitat of constant, extreme cold (-1.86 +/- 2 degrees C), and orthologs of the enzyme A4-lactate dehydrogenase (A4-LDH) in these species have adapted to this environment through higher catalytic rates, lower Arrhenius activation energies (Ea), and increases in the apparent Michaelis constant for the substrate pyruvate (Km(PYR)). Here, site-directed mutagenesis was used to determine which amino acid substitutions found in A4-LDH of the notothenioid Chaenocephalus aceratus, with respect to orthologs from warm-adapted teleosts, are responsible for these adaptive changes in enzyme function. Km(PYR) was measured in eight single and two double mutants, and Ea was tested in five single and two double mutants in the temperature range 0 degrees C-20 degrees C. Of the four mutants that had an effect on these parameters, two increased Ea but did not affect Km(PYR) (Gly224Ser, Ala310Pro), and two increased both Ea and Km(PYR) (Glu233Met, Gln317Val). The double mutants Glu233Met/Ala310Pro and Glu233Met/Gln317Val increased Km(PYR) and Ea to levels not significantly different from the A4-LDH of a warm temperate fish (Gillichthys mirabilis, habitat temperature 10 degrees C-35 degrees C). The four single mutants are associated with two alpha-helices that move during the catalytic cycle; those that affect Ea but not Km(PYR) are further from the active site than those that affect both parameters. These results provide evidence that (1) cold adaptation in A4-LDH involves changes in mobility of catalytically important molecular structures; (2) these changes may alter activation energy alone or activation energy and substrate affinity together; and (3) the extent to which these parameters are affected may depend on the location of the substitutions within the mobile alpha-helices, perhaps due to differences in proximity to the active site.     

Protein adaptations to temperature and pressure: complementary roles of adaptive changes in amino acid sequence and internal milieu

Retention of required structural and functional properties of proteins in species adapted to different temperatures and pressures is achieved through variation in amino acid sequence and accumulation of small organic solutes that stabilize protein traits. Conservation of ligand binding and catalytic rate can be achieved by minor differences in sequence. For orthologs of lactate dehydrogenase-A (A₄-LDH) temperature adaptation may involve only a single amino acid substitution. Adaptation involves changes in conformational mobility of regions of A₄-LDH that undergo movement during ligand binding, movements that are rate-limiting to catalysis. A model that integrates adaptations in sequence and intracellular milieu is developed on the basis of conformational microstates. Although orthologs of different thermally adapted species vary in stability, at physiological temperatures it is hypothesized that a similar ensemble of conformational microstates exists for all orthologs. Organic solutes stabilize this ensemble of microstates. Differences among orthologs in responses to organic solutes at a common temperature lead to similar responses at normal body temperatures. Because protein stability increases at high protein concentrations, intrinsic stabilities of proteins may reflect the protein concentrations of the cellular compartments in which they occur. Protein–stabilizing solutes like trimethylamine-N-oxide (TMAO) conserve protein function and structure at elevated hydrostatic pressures.     

Evolutionary convergence in adaptation of proteins to temperature: A4-lactate dehydrogenases of Pacific damselfishes (Chromis spp.)

We have compared the kinetic properties (Michaelis-Menten constant [K(m)] and catalytic rate constant [k(cat)]) and amino acid sequences of orthologs of lactate dehydrogenase-A (A(4)-LDH) from congeners of Pacific damselfishes (genus Chromis) native to cold-temperate and tropical habitats to elucidate mechanisms of enzymatic adaptation to temperature. Specifically, we determined whether the sites of adaptive variation and the types of amino acids involved in substitutions at these sites were similar in the Chromis orthologs and other orthologs of warm-adapted and cold-adapted A(4)-LDH previously studied. We report striking evolutionary convergence in temperature adaptation of this protein and present further support for the hypothesis that enzyme adaptation to temperature involves subtle amino acid changes at a few sites that affect the mobility of the portions of the enzyme that are involved in rate-determining catalytic conformational changes. We tested the predicted effects of differences in sequence using site-directed mutagenesis. A single amino acid substitution in a key hinge region of the A(4)-LDH molecule is sufficient to change the kinetic characteristics of a temperate A(4)-LDH to that of a tropical ortholog. This substitution is at the same location that was identified in previous studies of adaptive variation in A(4)-LDH and was hypothesized to be important in adjusting K(m) and k(cat). Our results suggest that certain sites within an enzyme, notably those that establish the energy changes associated with rate-limiting movements of protein structure during catalysis, are "hot spots" of adaptation and that common types of amino acid substitutions occur at these sites to adapt structural "flexibility" and kinetic properties. Thus, despite the wide array of options that proteins have to adjust their structural stabilities in the face of thermal stress, the adaptive changes that couple "flexibility" to alterations of function may be limited in their diversity.     

Effects of ribosomes and intracellular solutes on activities and stabilities of elongation factor 2 proteins from psychrotolerant and thermophilic methanogens

Low-temperature-adapted archaea are abundant in the environment, yet little is known about the thermal adaptation of their proteins. We have previously compared elongation factor 2 (EF-2) proteins from Antarctic (Methanococcoides burtonii) and thermophilic (Methanosarcina thermophila) methanogens and found that the M. burtonii EF-2 had greater intrinsic activity at low temperatures and lower thermal stability at high temperatures (T. Thomas and R. Cavicchioli, J. Bacteriol. 182:1328-1332, 2000). While the gross thermal properties correlated with growth temperature, the activity and stability profiles of the EF-2 proteins did not precisely match the optimal growth temperature of each organism. This indicated that intracellular components may affect the thermal characteristics of the EF-2 proteins, and in this study we examined the effects of ribosomes and intracellular solutes. At a high growth temperature the thermophile produced high levels of potassium glutamate, which, when assayed in vitro with EF-2, retarded thermal unfolding and increased catalytic efficiency. In contrast, for the Antarctic methanogen adaptation to growth at a low temperature did not involve the accumulation of stabilizing organic solutes but appeared to result from an increased affinity of EF-2 for GTP and high levels of EF-2 in the cell relative to its low growth rate. Furthermore, ribosomes greatly stimulated GTPase activity and moderately stabilized both EF-2 proteins. These findings illustrate the different physiological strategies that have evolved in two phylogenetically related but thermally distinct methanogens to enable EF-2 to function satisfactorily.     

Compatible solutes as protectants for zymogens against proteolysis

Compatible solutes are small organic osmoprotectants that have the capability to stabilize proteins. In coupled assays, the effect of the solutes ectoine, hydroxyectoine and betaine on the activation of the zymogens trypsinogen and chymotrypsinogen, catalyzed by enteropeptidase and trypsin, respectively, was studied. To different extents, all solutes protected the zymogens against activation. Ectoine (800 mM) was the most potent solute in reducing the formation of trypsin to 4% of the control value and of chymotrypsin to 23%. In separate experiments, the ability of the solutes to preserve proteolytic activity during incubation was investigated. After 4 h, trypsin and chymotrypsin completely lost their activity, but in the presence of ectoine, approximately 50% residual activity was maintained. It is proposed that a conformational shift of the protein towards folded, native-like states induced by preferential exclusion of the solute is responsible for the stabilizing and chaperone-like effects.     

Thermodynamics of the unfolding of the cold-shock protein from Thermotoga maritima.

Proteins from (hyper-)thermophiles are known to exhibit high intrinsic stabilities. Commonly, their thermodynamic characterization is impeded by irreversible side reactions of the thermal analysis or calorimetrical problems. Small single-domain proteins are suitable candidates to overcome these obstacles. Here, the thermodynamics of the thermal denaturation of the recombinant cold-shock protein (Csp) from the hyperthermophilic bacterium Thermotoga maritima (Tm) was studied by differential scanning calorimetry. The unfolding transition can be described over a broad pH range (3.5-8.5) by a reversible two-state process. Maximum stability (DeltaG (25 degrees C)=6.5 kcal/mol) was observed at pH 5-6 where Tm Csp unfolds with a melting temperature at 95 degrees C. The heat capacity difference between the native and the denatured states is 1.1(+/-0.1) kcal/(mol K). At pH 7, thermal denaturation occurs at 82 degrees C. The corresponding free energy profile has its maximum at 30 degrees C with DeltaGN-->U=4.8(+/-0.5) kcal/mol. At the optimal growth temperature of T. maritima (80 degrees C), Tm Csp in the absence of ligands is only marginally stable, with a free energy of stabilization not far beyond the thermal energy. With the known stabilizing effect of nucleic acids in mind, this suggests a highly dynamical interaction of Tm Csp with its target molecules.     

Dissociation-association of lactate dehydrogenase isozymes: influences on the formation of tetramers versus dimers of M4-LDH and H4-LDH

1. A molecular sieve membrane was used to separate active dimer vs active tetramer fractions of M4-LDH and H4-LDH. 2. Dissociation of both enzymes was influenced by enzyme protein concentration and by the concentration of added substrates, pyruvate or lactate. 3. Increasing lactate concentrations increased the fraction of tetrameric enzyme whereas increasing pyruvate (up to saturating levels) had the opposite effect, raising the content of dimer fraction. 4. For H4-LDH, levels of pyruvate that caused substrate inhibition reversed the effect of lower concentrations of pyruvate and reduced the dimer content. 5. The data suggest that dissociation-association of LDH may have functional importance, the dimer having a preferential role in pyruvate reduction and the tetramer a preferred function in lactate oxidation.     

Cold-adapted enzymes produced by fungi from terrestrial and marine Antarctic environments

Antarctica is the coldest, windiest, and driest continent on Earth. In this sense, microorganisms that inhabit Antarctica environments have to be adapted to harsh conditions. Fungal strains affiliated with Ascomycota and Basidiomycota phyla have been recovered from terrestrial and marine Antarctic samples. They have been used for the bioprospecting of molecules, such as enzymes. Many reports have shown that these microorganisms produce cold-adapted enzymes at low or mild temperatures, including hydrolases (e.g. α-amylase, cellulase, chitinase, glucosidase, invertase, lipase, pectinase, phytase, protease, subtilase, tannase, and xylanase) and oxidoreductases (laccase and superoxide dismutase). Most of these enzymes are extracellular and their production in the laboratory has been carried out mainly under submerged culture conditions. Several studies showed that the cold-adapted enzymes exhibit a wide range in optimal pH (1.0–9.0) and temperature (10.0–70.0?°C). A myriad of methods have been applied for cold-adapted enzyme purification, resulting in purification factors and yields ranging from 1.70 to 1568.00-fold and 0.60 to 86.20%, respectively. Additionally, some fungal cold-adapted enzymes have been cloned and expressed in host organisms. Considering the enzyme-producing ability of microorganisms and the properties of cold-adapted enzymes, fungi recovered from Antarctic environments could be a prolific genetic resource for biotechnological processes (industrial and environmental) carried out at low or mild temperatures.     

Lactate dehydrogenase from the Antarctic eelpout, Lycodichthys dearborni

As part of our studies to examine the molecular basis of cold-adaptation, we have determined the kinetic properties, thermal stability and deduced amino acid sequence of the enzyme lactate dehydrogenase (LDH) from an Antarctic zoarcid fish, Lycodichthys dearborni. Unlike Antarctic notothenioid fish which are endemic to the Southern Ocean, zoarcid fish are cosmopolitan and have a substantially longer evolutionary history as a sub-order. The A₄-LDH isoform was isolated and purified from the white muscle of L. dearborni. The kinetic parameters K_m^PYR and k_cat were determined at temperatures from 0 to 25°C. K_m^PYR was substantially higher at low temperatures than those from Antarctic and temperate notothenioid fish, whereas k_cat at these temperatures was essentially the same as those of the other fish LDH in this study. The sequence of L. dearborni A₄-LDH was determined from cDNA derived from white muscle RNA and found to be similar to, but distinct from, the A₄-LDH sequences of Antarctic notothenioid fish. Molecular modelling based on the structure of the A₄-LDH from Pagothenia borchgrevinki suggested that three conservative amino acid changes within the core of the protein that are not directly part of the active site but which might nonetheless influence the active site, may be important in cold-adaptation in L. dearborni A₄-LDH, and that several other changes on the surface of the protein might also play a role in cold-adaptation.     

Compatible solute effects on thermostability of glutamine synthetase and aspartate transcarbamoylase from Methanococcus jannaschii

Methanococcus jannaschii accumulates alpha- and beta-glutamate as osmolytes. The effect of these and other solutes on the thermostability of two multisubunit metabolic enzymes from M. jannaschii, aspartate transcarbamoylase catalytic trimer (ATCase C3) and glutamine synthetase (GS), has been measured and compared to solute effects on bacterial mesophilic counterparts in order to explore if osmolytes accumulated by each organism can preferentially stabilize the proteins to thermal unfolding. For both ATCase enzymes and for the B. subtilis GS, the solutes normally accumulated by the organism were very effective in protecting the enzyme from losing activity at high temperatures, although solute effects on loss of secondary structure did not necessarily correlate with this thermoprotection of activity. The recombinant M. jannaschii GS exhibited quite different behavior. The pure enzyme had a thermal unfolding transition with a midpoint temperature (Tm) less than 60 degrees C, well under the growth temperature of the organism (85 degrees C). None of the small molecule solutes tested (including the K+-glutamate isomers accumulated by M. jannaschii) significantly stabilized the protein to incubation at 85 degrees C. Instead, protein-protein interactions, as illustrated by E. coli GroEL or ribosomal protein L2 stabilization of GS, appeared to be the dominant factor in stabilizing this archaeal enzyme at the growth temperature.     

Increased thermal stability of proteins in the presence of naturally occurring osmolytes.

Organisms and cellular systems which have adapted to stresses such as high temperature, desiccation, and urea-concentrating environments have responded by concentrating particular organic solutes known as osmolytes. These osmolytes are believed to confer protection to enzyme and other macromolecular systems against such denaturing stresses. Differential scanning calorimetric (DSC) experiments were performed on ribonuclease A and hen egg white lysozyme in the presence of varying concentrations of the osmolytes glycine, sarcosine, N,N-dimethylglycine, and betaine. Solutions containing up to several molar concentrations of these solutes were found to result in considerable increases in the thermal unfolding transition temperature (Tm) for these proteins. DSC scans of ribonuclease A in the presence of up to 8.2 M sarcosine resulted in reversible two-state unfolding transitions with Tm increases of up to 22 degrees C and unfolding enthalpy changes which were independent of Tm. On the basis of the thermodynamic parameters observed, 8.2 M sarcosine results in a stabilization free energy increase of 7.2 kcal/mol for ribonuclease A at 65 degrees C. This translates into more than a 45,000-fold increase in stability of the native form of ribonuclease A over that in the absence of sarcosine at this temperature. Catalytic activity measurements in the presence of 4 M sarcosine give kcat and Km values that are largely unchanged from those in the absence of sarcosine. DSC of lysozyme unfolding in the presence of these osmolytes also results in Tm increases of up to 23 degrees C; however, significant irreversibly occurs with this protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Seasonal Changes in the Content of Urotensin II in the Urophysis of the Goby,Gillichthys mirabilis

A seasonal study of urotensin II content of the urophysis of the goby, Gillichthys mirabilis. was conducted from March 1979 to June 1980 in relation to certain internal and environmental changes. Urotensin II content (lowest in November–January) is inversely correlated with female gonadosomatic index and to some extent with rainfall (and hence dilution of the environmental salinity). In addition, there appears to be a direct correlation between UII content and daylength and temperature.     

Mechanism of solvent induced thermal stabilization of papain

In the present study an attempt is made to elucidate the effects of various cosolvents, such as sorbitol, sucrose, xylose and glycerol, on papain. The stabilizing effects of these cosolvents on the structure and function of papain is determined by the activity measurements, fluorescence spectroscopy and differential scanning calorimetry (DSC). The enzyme activity measurements indicate several fold increase in the thermal stability of the enzyme in all the cosolvents used. The thermal denaturation studies of papain in presence of various concentrations of cosolvents indicated a shift in the apparent thermal denaturation temperature (app Tm) suggesting increased thermal stability of papain in presence of cosolvents. The app Tm shifted from a control value of 83+/-1 degrees C to a value of >90+/-1 degrees C in presence of 40% sorbitol. The DSC thermogram for native papain can be clearly deconvoluted into two transitions corresponding to left and right domain and in presence of cosolvents both transitions A and B shift to higher temperature. Maximum stabilization was seen in case of 30% sorbitol where the thermal transition temperatures increased compared to control. The results from partial specific volume measurements of papain in presence of cosolvents suggest that the preferential interaction parameter (xi3) was negative in all cosolvents and maximum hydration was observed in the case of glycerol where the preferential interaction parameter was 0.165g/g. These above results suggest that there is a considerable increase in the thermal stability of papain in presence of these cosolvents as a result of preferential hydration.     

Lipolytic activity of Antarctic cold-adapted marine bacteria (Terra Nova Bay, Ross Sea)

AIMS: The aim of this study was to investigate the lipolytic activity of cold-adapted Antarctic marine bacteria and, furthermore, the combined effect of some environmental factors on this enzymatic process. METHODS AND RESULTS: Strains were assayed for lipolytic activity on a basal medium amended with seven individual fatty acid esters. A significant activity was observed for 148 isolates (95.5% of the total screened). The interactive effect of pH, temperature and NaCl concentration on the substrates was tested for six representative isolates, identified as Pseudoalteromonas, Psychrobacter and Vibrio. Differences between strains according to NaCl and pH tolerances were observed. Only one strain degraded the substrate more efficiently at 4 degrees C than at 15 degrees C. CONCLUSIONS: Our findings demonstrate that the lipolytic activity of Antarctic marine bacteria is rather variable, depending on culture conditions, and occurs in a wide range of salt concentration and pH. SIGNIFICANCE AND IMPACT OF THE STUDY: Isolation and characterization of bacteria that are able to efficiently remove lipids at low temperatures will provide insight into the possibility to use cold-adapted bacteria as a source of exploitable enzymes. Moreover, research on the interactive effects of salt concentration, pH and temperature will be useful to understand the true enzyme potentialities for industrial applications.     

Preferential binding of two compatible solutes to the glycan moieties of Peniophora lycii phytase

Regulation of hydration behavior, and the concomitant effects on solubility and other properties, has been suggested as a main function of protein glycosylation. In this work, we have studied the hydration of the heavily glycosylated Peniophora lycii phytase in solutions (0.15-1.1 m) of the two compatible solutes glycerol and sorbitol. Osmometric measurements showed that glycerol preferentially binds to phytase (i.e., glycerol-glycoprotein interactions are more favorable than water-glycoprotein interactions resulting in a preferential accumulation of glycerol near the protein interface), while sorbitol is preferentially excluded from the hydration sphere (water-glycoprotein interactions are the more favorable). To assess contributions from carbohydrate and peptide moieties, respectively, we compared phytase (Phy) and a modified, yet enzymatically active form (dgPhy) in which 90% of the glycans had been removed. This revealed that both polyols showed a pronounced and approximately equal degree of preferential binding to the carbohydrate moiety. This preferential binding of polyols to glycans is in contrast to the exclusion from peptide interfaces observed here (for dgPhy) and in numerous previous reports on nonglycosylated proteins. Despite the distinct differences between peptide and carbohydrate groups, glycosylation had no effect on the stabilizing action provided by glycerol and sorbitol. On the basis of this, it was concluded that the carbohydrate mantle of Phy is equally accessible in the native and thermally denatured states, respectively (most likely fully accessible in both), and thus that its interactions with compatible solutes have little or no effect on conformational equilibria of the glycoprotein. For solubility and aggregation equilibria, on the other hand, the results suggest a polyol-induced stabilization of monomeric forms.     

Mechanism of the stabilization of ribonuclease A by sorbitol: preferential hydration is greater for the denatured then for the native protein.

The effect of interactions of sorbitol with ribonuclease A (RNase A) and the resulting stabilization of structure was examined in parallel thermal unfolding and preferential binding studies with the application of multicomponent thermodynamic theory. The protein was stabilized by sorbitol both at pH 2.0 and pH 5.5 as the transition temperature, Tm, was increased. The enthalpy of the thermal denaturation had a small dependence on sorbitol concentration, which was reflected in the values of the standard free energy change of denaturation, delta delta G(o) = delta G(o) (sorbitol) - delta G(o)(water). Measurements of preferential interactions at 48 degrees C at pH 5.5, where protein is native, and pH 2.0 where it is denatured, showed that sorbitol is preferentially excluded from the denatured protein up to 40%, but becomes preferentially bound to native protein above 20% sorbitol. The chemical potential change on transferring the denatured RNase A from water to sorbitol solution is larger than that for the native protein, delta mu(2D) > delta mu(2N), which is consistent with the effect of sorbitol on the free energy change of denaturation. The conformity of these results to the thermodynamic expression of the effect of a co-solvent on denaturation, delta G(o)(W) + delta mu(D)(2)delta G(o)(S) + delta mu(2D), indicates that the stabilization of the protein by sorbitol can be fully accounted for by weak thermodynamic interactions at the protein surface that involve water reversible co-solvent exchange at thermodynamically non-neutral sites. The protein structure stabilizing action of sorbitol is driven by stronger exclusion from the unfolded protein than from the native structure.     

Gene amplification and cold adaptation of pepsin in Antarctic fish. A possible strategy for food digestion at low temperature

Cold-adapted organisms have developed a number of adjustments at the molecular level to maintain metabolic functions at low temperatures. Among other features, they can produce enzymes characterized by a high turnover number or a high catalytic efficiency. The present work is aimed at investigating the process of food digestion at low temperature through the study of pepsins in Antarctic notothenioids. For such a purpose, we have cloned and sequenced three forms of pepsin A and a single form of gastricsin from the gastric mucosa of Trematomus bernacchii (rock cod). Phylogenetic analysis has suggested that the three pepsin A isotypes arose from two gene duplication events leading to the most ancestral pepsin A3 and to the most recent forms represented by pepsin A1 and pepsin A2. Molecular modeling has unraveled significant structural differences in these enzymes with respect to their mesophilic counterparts. Hydropathy and flexibility determined on the substrate-binding subsites of Antarctic and mesophilic pepsins have shown for pepsin A2 reduced hydropathy and increased flexibility at the level of the substrate cleft, features typical of cold-adapted enzymes. Northern blot analysis of RNA from rock cod gastric mucosa hybridized with molecular probes designed on specific regions of different pepsin forms has shown that rock cod pepsin genes are expressed at comparable levels. The present results suggest that the Antarctic rock cod adopted two different strategies to accomplish efficient protein digestion at low temperature. One mechanism is the gene duplication that increases enzyme production to compensate for the reduced kinetic efficiency, the other is the expression of a new enzyme provided with features typical of cold-adapted enzymes.     

Modulation of the multistate folding of designed TPR proteins through intrinsic and extrinsic factors

Tetratricopeptide repeats (TPRs) are a class of all alpha-helical repeat proteins that are comprised of 34-aa helix-turn-helix motifs. These stack together to form nonglobular structures that are stabilized by short-range interactions from residues close in primary sequence. Unlike globular proteins, they have few, if any, long-range nonlocal stabilizing interactions. Several studies on designed TPR proteins have shown that this modular structure is reflected in their folding, that is, modular multistate folding is observed as opposed to two-state folding. Here we show that TPR multistate folding can be suppressed to approximate two-state folding through modulation of intrinsic stability or extrinsic environmental variables. This modulation was investigated by comparing the thermodynamic unfolding under differing buffer regimes of two distinct series of consensus-designed TPR proteins, which possess different intrinsic stabilities. A total of nine proteins of differing sizes and differing consensus TPR motifs were each thermally and chemically denatured and their unfolding monitored using differential scanning calorimetry (DSC) and CD/fluorescence, respectively. Analyses of both the DSC and chemical denaturation data show that reducing the total stability of each protein and repeat units leads to observable two-state unfolding. These data highlight the intimate link between global and intrinsic repeat stability that governs whether folding proceeds by an observably two-state mechanism, or whether partial unfolding yields stable intermediate structures which retain sufficient stability to be populated at equilibrium.     

Temperature adaptation of DNA ligases from psychrophilic organisms

DNA ligases operating at low temperatures have potential advantages for use in biotechnological applications. For this reason, we have characterized the temperature optima and thermal stabilities of three minimal Lig E-type ATP-dependent DNA ligase originating from Gram-negative obligate psychrophilic bacteria. The three ligases, denoted Vib-Lig, Psy-Lig, and Par-Lig, show a remarkable range of thermal stabilities and optima, with the first bearing all the hallmarks of a genuinely cold-adapted enzyme, while the latter two have activity and stability profiles more typical of mesophilic proteins. A comparative approach based on sequence comparison and homology modeling indicates that the cold-adapted features of Vib-Lig may be ascribed to differences in surface charge rather than increased local or global flexibility which is consistent with the contemporary emerging paradigm of the physical basis of cold adaptation of enzymes.