De Cuyper M., Bulte J.W.M. - Physics and chemistry basis of biotechnology (Vol. 7) (2002)(en)

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V 196F, engineering a salt bridge (N 1 50D) and an aromatic interaction (V 196F), caused decreased activity and significantly increased melting temperature (44°C to 46.4°C), establishing a correlation between increased stability and lowered activity (D'Amico et al., personal communication). Some other site-directed mutagenesis experiments have been carried on subtilisin [45] and a moderately stable thermolysin like protease [76]. Both studies revealed that the stability of the mutated enzymes was drastically improved due to a small number of mutations, while increasing or retaining the original catalytic properties of the enzymes, which does not correspond with the stabilityspecific activity-flexibility assumption. Moreover, a recent in vitro evolution experiment has proved that stability and catalytic activity are not systematically inversely related, since random mutagenesis on the Bacillus subtilis p-nitrobenzyl esterase led to an increase in stability (>14°C increase in Tm) without compromising catalytic activity [77]. In fact, reduced stability may not necessarily arise from a general reduction in strength of intramolecular forces, but from weakened interactions in one or a few important regions of the structure [78].

Figure 1. Thermal unfolding of the psychrophilic Pseudomonas sp. TACII 18 (left and yeast (right) phosphoglycerate kinase (PGK). a to d: baseline-subtracted thermograms recorded by DSC. a and b: free enzymes (the deconvolution in :wo transitions is dotted). c and d: PGK in the presence of 5 mM 3-phosphoglyceric acid and 5 mM Mg-ADP (solid lines). In c and d, the thermogram for free enzyme is given as a dashed line for comparison. HLd, heat-labile domain; HSd, heat-stable domain.

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Some recent works have corroborated this statement. In the case of citrate synthase [79], a calculation of crystallographic temperature (B) factors, which, to a certain extent, reflect the flexibility or disorder of the crystal structure in a given region, revealed an unexpected higher flexibility for the thermophilic enzyme when considering the overall protein structure. However, in the psychrophilic homologue, the small domain showed a higher degree of flexibility than did the large domain. This inequality could promote activity at low temperatures, since a precise positioning of the small domain following substrate binding is necessary for efficient catalysis. As mentioned by Fields and Somero [80], notothenioid A4-lactate dehydrogenase has adapted to cold by increasing flexibility of small areas of the molecule which affect the mobility of adjacent active-site structures. The increased flexibility may reduce energy barriers to rate-governing shifts in conformation and thereby, increase kcat. Moreover, crystallisation of malate dehydrogenase from the Arctic Aquaspirillium arcticum revealed that the active site of this enzyme is more flexible than that of the thermophilic one, facilitating efficient catalysis at low temperatures [81]. Finally, differential scanning calorimetry performed on the phosphoglycerate kinase from the Antarctic Pseudomonas sp. TACII 18 [60] revealed unusual variations of its conformational stability in free (Figure la) and liganted forms (Figure 1c). As shown in Figure la, the psychrophilic PGK is characterised by a heat-labile (HLd) and a heat-stable (HSd) domain, whereas its mesophilic homologue has two domains unfolding simultaneously. In the substrate-free form of psychrophilic enzyme (Figure la), the heat-labile and the thermostable domain unfold independently. The existence of a stabilising domain has previously been mentioned in xylanases [82], in which the increased stability of one domain promotes the stability of the whole molecule. Similarly, Bentahir and his colleagues have proposed that the PGK heat labile domain acts as a destabilising domain, providing the required flexibility around the active site for catalysis at low temperatures. Thus cold adapted proteins have evolved, either by displaying a reduced stability of all calorimetric units giving rise to native states of the lowest stability [83] or by being constituted of different elements, some controlling protein stability and others conferring the required flexibility for efficient catalysis at the habitat temperature.

6. Structural comparisons

The thermostability of thermophilic enzymes has been extensively investigated and several possible determinants of this stability have been proposed [84]. In contrast, structural comparisons of cold active enzymes with their mesophilic and thermophilic counterparts have been limited, until recently, to the analysis of homology models and/or sequence alignments for a limited number of enzymes from bacteria, yeast and fish, such as α-amylase [37, 85], triosephosphate isomerase [54], subtilisin [44], trypsin (fish) [86], β-lactamase [38], elastase (fish) [87], 3-isopropylmalate dehydrogenase [88], lipase [41], elongation factor (EF) 2 [89] and EF-G [90], xylanase (yeast) [48] and phosphoglycerate kinase [60]. These studies have revealed that only subtle modifications of the enzyme conformation account for low stability and that the

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adaptation strategy is unique to each enzyme. Moreover neither the amino-acid residues involved in the catalytic process nor the topology of the active site of the enzymes are affected to any extent in cold-adapted proteins. This suggests that cold-adaptation does not involve new catalytic mechanisms, but rather that conventional mechanisms are modified to operate better at low temperatures. In fact, these works have revealed that the main features possibly implicated in cold-adaptation consisted of fewer salt links (especially with a reduced number of arginine residues), hydrogen bonds, isoleucine clusters and proline residues in loops, extended and highly charged surface loops, an increased number of glycine and serine residues close to functional motifs, a reduction in the hydrophobicity of the enzyme and an increase in the number of interactions between the enzyme surface and the solvent. All these parameters have been reviewed by Feller et al. [33, 91] and Gerday et al. [34]. Interestingly, the same structural factors have been implicated in the stability of thermophilic proteins [62, 92-96] suggesting that there is a continuum in the strategy of protein adaptation to temperature.

The putative parameters involved in cold adaptation have been confirmed by recent resolutions of crystallographic 3D structures of some cold enzymes: salmon trypsin [78], α-amylase from Alteromonas haloplanktis [97-99], Ca2+-Zn2+ protease from Pseudomonas aeruginosa [100], triosephosphate isomerase from Vibrio marinus [55], citrate synthase from the Antarctic bacterial strain DS2-R [79] and malate dehydrogenase from Aquaspirillium arcticum [8 1]. For instance, these enzymes frequently display a weakening of the intramolecular interactions. However, an increase in intramolecular ion pairs has been observed in the heat-labile citrate synthase, demonstrating that, as previously mentioned, all psychrophilic enzymes do not use the same strategy to increase structural resilience. The authors explained that this increase may serve to prevent cold denaturation by counteracting the reduced thermodynamic stability originating from an increase in solvent-hydrophobic interactions. Exposure of hydrophobic residues to solvent is indeed destabilising, due to the ordering of water molecules. Therefore, the elimination of destabilising hydrophobic interactions with solvent may be necessary for hyperthermostability, whereas their presence in thermolabile enzymes may be an important factor in preserving the protein structure at low temperatures. Increased solvent-hydrophobic residues interaction has been reported for salmon trypsin and a cold-active a-amylase. Decreased stability of cold-enzymes is also reached by a general weakening of interdomain or intersubunit interactions, which emerges as a critical force in stabilising hyperthermophilic enzymes [95, 101, 102]. As observed for salmon trypsin, citrate synthase and malate dehydrogenase, a precise distribution of surface charges favours a better electrostatic attraction of substrates, contributing to an increased catalytic efficiency. Finally a better accessibility of the catalytic cavity can improve substrate accommodation; this has been described for salmon trypsin [78], a-amylase (Figure 2) [103] and citrate synthase [79] and could lead to a higher specific activity at low temperatures. All the above-mentioned factors may possibly improve the overall or local flexibility of the molecular edifice.

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Figure 2. Active site accessibility. Upper panel: superimposition of the variable loops bordering the active site of the psychrophilic a-amylase from Pseudoalteromonas haloplanktis (backbone in black) and ofpigpancreatic a-amylase (backbone in grey). The carbohydrate inhibitor acarbose bound to the active site is also shown (centre of the figure, in black). These variable loops are markedly shorter in the cold-active enzyme. Lower panel: tangential view of the molecular surfaces facing the external medium (upper side). Acarbose is removed for clarity. The loops around the catalytic cleft of the coldactive enzyme are less pro truding and favour active site accessibility. Picture generated by Swiss-PDBViewer [I27] using data from references [98, 99].

7. Fundamental and biotechnological applications

It has already been demonstrated that cold-evolved enzymes constitute useful tools for fundamental studies in protein folding. Moreover, psychrophilic organisms and their products offer a high potential for biotechnological applications [15, 104, 105]. For example, a well known and important application of cold enzymes, such as proteases, lipases, α-amylases and cellulases, is their use in the detergent industry. Indeed, cold washing allows energy savings and a reduction in wear and tear. However, the thermal instability and storage of these enzymes can constitute a considerable drawback. It is, though, always possible to engineer recombinant enzymes in which reasonable stability is coupled with high catalytic efficiency [45]. In the textile industry, the use of a cold-

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adapted cellulase would offer an excellent alternative to stone-washing of jeans and biopolishing of cotton fibres [ 106]. Pre-treatment of tissues with cellulases, under the appropriate conditions, would reduce pill-formation (due to cotton fibre ends protruding from the main tissue fibres) and increase both the durability and the softness of the tissue. The current treatment, however, leads to an alteration of the main fibre, as a result of the resistance of the mesophilic enzyme to heat-inactivation. The use of psychrophilic cellulase, which is easily inactivated, would allow circumventing this problem. One can also take advantage of the high thermosensitivity of psychrophilic enzymes in the food industry. The possible applications of cold-evolved enzymes are numerous. The main goals include the improvement of taste and product quality as well as the minimisation of mesophilic microorganism contamination. In order to prevent early degradation due to microbial growth, low temperature is often mandatory in the food industry. Thus, properties of psychrophilic enzymes, i.e. high specific activity at low and moderate temperatures and low thermal stability, would allow food to retain their freshness and flavour during the enzymatic reaction and following enzyme inactivation. Hence enzymes are promising in various fields such as lipid extraction , processing of fruit juices (pectinases) and cheese, modification of food texture, improvement in the quality of milk (b-galactosidases), bread (amylases, proteases, xylanases) and alcoholic drinks.

Some cold-evolved enzymes, such as DNA ligase, alkaline phosphatase and uracilDNA glycosylase, also constitute new and performing tools in molecular biology. DNA ligases are essential in molecular biology. In fact, the ligation reaction requires a low temperature, in order to ensure sufficient temporal base pairing (through hydrogen bonding) to allow the formation of a phosphodiester bond. Commercially mesophilic DNA ligases have, however, relatively poor activity at temperatures below 15°C and require long incubation times. Under such conditions, the action of residual nucleases is favoured, which can interfere with the ligation reaction. Pseudoalteromonas haloplanktis DNA ligase displays a high catalytic efficiency at low and moderate temperatures, compared to its mesophilic counterpart [61]. In addition, a relatively low inactivation temperature can be used, which does not denature DNA, and therefore, this enzyme represents a novel tool in biotechnology. Radioactive end-labelling of nucleic acids by T4 polynucleotide kinase requires the removal of the existing phosphates at the 5’ ends of DNA by alkaline phosphatase (APase). This enzyme must be inactivated after the reaction, to prevent degradation of labelled ATP and the loss of label from the substrates. However, known mesophilic APases display great thermal stability, leading to incomplete enzyme inactivation and interference with subsequent kinase and ligase reaction. Hence the remarkable temperature sensitivity of psychrophilic HK47 APase appears to be a useful feature for 5’ end-labelling [107]. Uracil-DNA glycosylase (UNG), produced by a psychrophilic marine bacterium, is also a potential tool for the biotechnology market [108]. UNG belong to a specific class of DNA repair enzymes [109]. These enzymes are mainly used to prevent carryover contamination in polymerase chain reaction (PCR), and hence avoiding false positive results, due to the contamination of PCR samples by products from previous amplifications. Prior to the actual PCR, the enzyme must be heat-inactivated. However, E. coli UNG, usually used,

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is not completely inactivated during heat treatment, leading to degradation of the newly synthesised PCR product. This problem can be overcome by using the heat-labile UNG [108]. We have also to emphasise that the development of a ‘cold’ gene expression system is under investigation [110-1 12]. In fact, since cold-active enzymes are usually thermolabile, ordinary expression systems such as the E. coli expression system, may not be the most suitable for the expression of cold-active enzymes. So far, some coldshock proteins have been well characterised [26]. CspA is the major cold-shock protein in E. coli and recently, an expression system with a cspA promoter, which is controlled by cold-shock treatment, has been developed [113]. Furthermore, other cold-inducible promoters in E. coli have also been reported [ 114]. Therefore, the availability of such a cold-shock inducible expression system could enhance the expression yield by minimising proteolytic degradation as well as the accumulation of the recombinant product in insoluble precipitates. In addition, Remaut et al. [112] have developed an expression system in which E. coli -derived expression controlling elements are introduced into psychrophilic hosts, using as vector system a broad-host-range plasmid. It was shown that E. coli lacIq-Ptrc repressor-promoter system is operative at temperatures as low as 4°C in two different psychrotrophic species.

Another interesting aspect of some Antarctic microorganisms is their production of polyunsaturated fatty acids (PUFAs), including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (for review, see [115, 116]). In fact, they attract considerable attention as sources of pharmaceutical agents, functional foods, and supplement nutrition. Among other, PUFAs are essential for normal growth and development of the larvae of many aquaculture species. While bacteria have previously been considered for use in aquaculture feeds, their lack of essential PUFA was seen as a major drawback [1 17]. However, it now appears that some strains of Antarctic bacteria also produce high levels of PUFA [118, 119]. Use of such PUFA-producing microheterotrophs in aquaculture diets, livestock and human diets is now an expanding area of interest.

Over the past decade numerous environments and especially low temperature habitats have been contaminated by oils, which has led to the investigation of hydrocarbon degradation by Antarctic micro-organisms [ 120122]. Degradation of xenobiotic compounds including diesel oil and polychlorinated biphenyls by psychrophilic bacteria could offer a possible alternative to physicochemical methods. Moreover, the addition of such bacteria to contaminated cold area should help to enhance the biodegradation ofhydrocarbons [18, 123-126].

8. Conclusions

In the last few years, increased attention has been focused on enzymes produced by cold-adapted micro-organisms. It has emerged that psychrophilic enzymes represent an extremely powerful tool in both protein folding investigations and for biotechnological purposes. Such enzymes are characterised by an increased thermosensitivity and, most of them, by a higher catalytic efficiency at low and moderate temperatures, when compared to their mesophilic counterparts. The high thermosensitivity probably

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originates from an increased flexibility of either a selected area of the molecular edifice or the overall protein structure, providing enhanced abilities to undergo conformational changes during catalysis at low temperatures. Structure modelling and recent crystallographic data have allowed to elucidate the structural parameters that could be involved in this higher resilience. It was demonstrated that each psychrophilic enzyme adopts its own adaptive strategy. It appears, moreover, that there is a continuum in the strategy of protein adaptation to temperature, as the previously mentioned structural parameters are implicated in the stability of thermophilic proteins. Additional 3D crystal structures, site-directed and random mutagenesis experiments should now be undertaken to further investigate the stability-flexibility-activity relationship,

Acknowledgements

We are grateful to N. Gerardin and R. Marchand for advises technical assistance. We acknowledge the "Institut Français de Recherche et de Technologie polaire" for generously accommodating our research fellows at the French Antarctic Station J.S. Dumont d'Urville in Terre Adélie.

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