Approaches for deciphering the structural basis of low temperature enzyme activity

An increasing number of enzymes active at low temperature are being studied to help determine the structural features important for cold-activity. This review examines the diversity of prokaryotic cold-active enzymes and the features proposed to account for low temperature activity. We then consider the difficulty of identifying the key structural features needed for cold-activity and the need to compare enzymes having different temperature optima from phylogenetically related organisms to determine features responsible for low temperature activity. In addition to studying naturally occurring enzymes, directed evolution experiments are discussed as methods for examining the proposed mechanisms influencing the thermal dependence of activity.     

Some like it cold: biocatalysis at low temperatures

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.     

Psychrophilic yeasts from worldwide glacial habitats: diversity, adaptation strategies and biotechnological potential

Glacial habitats (cryosphere) include some of the largest unexplored and extreme biospheres on Earth. These habitats harbor a wide diversity of psychrophilic prokaryotic and eukaryotic microorganisms. These highly specialized microorganisms have developed adaptation strategies to overcome the direct and indirect life-endangering influence of low temperatures. For many years Antarctica has been the geographic area preferred by microbiologists for studying the diversity of psychrophilic microorganisms (including yeasts). However, there have been an increasing number of studies on psychrophilic yeasts sharing the non-Antarctic cryosphere. The present paper provides an overview of the distribution and adaptation strategies of psychrophilic yeasts worldwide. Attention is also focused on their biotechnological potential, especially on their exploitation as a source of cold-active enzymes and for bioremediation purposes.     

Molecular basis of cold adaptation

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.     

Protein flexibility in psychrophilic and mesophilic trypsins. Evidence of evolutionary conservation of protein dynamics in trypsin-like serine-proteases

To shed light on the molecular features related to cold-adaptation in serine-proteases, we have carried out molecular dynamics simulations of homologous mesophilic and psychrophilic trypsins, with particular attention to evaluation of intramolecular interactions and flexibility. Psychrophilic trypsins present fewer interdomain interactions and enhanced localized flexibility in regions close to the catalytic site. Notably, these regions fit well with the pattern of protein flexibility previously reported for psychrophilic elastases. Our results indicate that specific sites within the serine-protease fold can be considered hot spots of cold-adaptation and that psychrophilic trypsins and elastases have independently discovered similar molecular strategies to optimize flexibility at low temperatures.     

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.

     

Structural features of cold-adapted dimeric GH2 β-D-galactosidase from Arthrobacter sp. 32cB

Crystal structures of cold-adapted β-d-galactosidase (EC 3.2.1.23) from the Antarctic bacterium Arthrobacter sp. 32cB (ArthβDG) have been determined in an unliganded form resulting from diffraction experiments conducted at 100 K (at resolution 1.8 Å) and at room temperature (at resolution 3.0 Å). A detailed comparison of those two structures of the same enzyme was performed in order to estimate differences in their molecular flexibility and rigidity and to study structural rationalization for the cold-adaptation of the investigated enzyme. Furthermore, a comparative analysis with structures of homologous enzymes from psychrophilic, mesophilic, and thermophilic sources has been discussed to elucidate the relationship between structure and cold-adaptation in a wider context. The performed studies confirm that the structure of cold-adapted ArthβDG maintains balance between molecular stability and structural flexibility, which can be observed independently on the temperature of conducted X-ray diffraction experiments. Obtained information about proper protein function under given conditions provide a guideline for rational engineering of proteins in terms of their temperature optimum and thermal stability.     

Proteins of higher fungi--from forest to application

Mushrooms are rapidly becoming recognized as a promising source of novel proteins. Several proteins showing unique features have been isolated, including lectins, lignocellulolytic enzymes, protease inhibitors and hydrophobins. They can offer solutions to several medical and biotechnological problems such as microbial drug resistance, low crop yields, and demands for renewable energy. Large-scale production and industrial application of some fungal proteins proves their biotechnological potential and establishes higher fungi as a valuable, although relatively unexplored, source of unique proteins. This review provides the first comprehensive overview of known proteins from mushrooms, describes the process of acquiring a new bioactive protein, and provides an overview of current and anticipated applications of these proteins across biotechnology, medicine and agriculture.     

Backbone and side chain 1H, 15N and 13C assignments for a thiol-disulphide oxidoreductase from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125

Enzymes produced by psychrophilic organisms have successfully overcome the low temperature challenge and evolved to maintain high catalytic rates in their permanently cold environments. As an initial step in our attempt to elucidate the cold-adaptation strategies used by these enzymes we report here the ¹H, ¹⁵N and ¹³C assignments for the reduced form of a thiol-disulphide oxidoreductase from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125.     

Crystal structure of a cold-adapted class C beta-lactamase

In this study, the crystal structure of a class C beta-lactamase from a psychrophilic organism, Pseudomonas fluorescens, has been refined to 2.2 A resolution. It is one of the few solved crystal structures of psychrophilic proteins. The structure was compared with those of homologous mesophilic enzymes and of another, modeled, psychrophilic protein. The elucidation of the 3D structure of this enzyme provides additional insights into the features involved in cold adaptation. Structure comparison of the psychrophilic and mesophilic beta-lactamases shows that electrostatics seems to play a major role in low-temperature adaptation, with a lower total number of ionic interactions for cold enzymes. The psychrophilic enzymes are also characterized by a decreased number of hydrogen bonds, a lower content of prolines, and a lower percentage of arginines in comparison with lysines. All these features make the structure more flexible so that the enzyme can behave as an efficient catalyst at low temperatures.     

Toward a molecular understanding of cold activity of enzymes from psychrophiles

Despite the fact that a much greater proportion of the earth environment is cold rather than hot, much less is known about psychrophilic, cold-adapted microorganisms compared with thermophiles living at high temperatures. In particular, investigation of the molecular basis of cold-active enzymes from psychrophiles has only recently received concerted research attention, in measure as a result of the EC-funded project COLDZYME. This research effort has been stimulated by the realization that such cold-active enzymes offer novel opportunities for biotechnological exploitation. Only very recently has the first cold-active enzyme, α-amylase, been crystallized, and this success was followed rapidly by others. This effort has facilitated a direct approach to solving the three-dimensional structure of cold-active enzymes to complement the gene homology modeling that had been performed previously. Recently studies have highlighted how different adaptations are used by different enzymes to achieve conformational flexibility at low temperatures, and how such adaptations are not necessarily the opposite of those that confer thermostability to proteins in thermophilic counterparts. This review also highlights initial successes in engineering genetically improved thermal stability in cold-active enzymes to give improved catalysts for low-temperature biotechnology. Received: July 11, 1999 / Accepted: December 27, 1999     

Stepwise adaptations to low temperature as revealed by multiple mutants of psychrophilic α-amylase from Antarctic Bacterium

The mutants Mut5 and Mut5CC from a psychrophilic α-amylase bear representative stabilizing interactions found in the heat-stable porcine pancreatic α-amylase but lacking in the cold-active enzyme from an Antarctic bacterium. From an evolutionary perspective, these mutants can be regarded as structural intermediates between the psychrophilic and the mesophilic enzymes. We found that these engineered interactions improve all the investigated parameters related to protein stability as follows: compactness; kinetically driven stability; thermodynamic stability; resistance toward chemical denaturation, and the kinetics of unfolding/refolding. Concomitantly to this improved stability, both mutants have lost the kinetic optimization to low temperature activity displayed by the parent psychrophilic enzyme. These results provide strong experimental support to the hypothesis assuming that the disappearance of stabilizing interactions in psychrophilic enzymes increases the amplitude of concerted motions required by catalysis and the dynamics of active site residues at low temperature, leading to a higher activity.     

A recombinant subtilisin with keratinolytic and fibrin(ogen)olytic activity

KerS14 is a keratinase with great potential in tannery, since it degrades keratin without damaging collagen, a feature suitable for various industrial uses. The enzyme was previously characterized and described as a serino endopeptidase belonging to the subtilisin group. However, KerS14 low thermal stability impairs its biotechnological potential. The present work presents several attempts to improve KerS14 thermal stability. KerS14 ORF was cloned into pET-5a vector with a His-tag at C-terminal, and four different mutants enzymes (G61C, S98C, P239R and G61C-S98C) were produced by site-direct mutagenesis. The recombinant enzyme and four mutants were expressed, purified and characterized regarding their thermal stability, optimum temperature and pH. The presence of a His-tag was shown to increase the KerS14 thermal stability, and to decrease the thermal stability of mutant enzymes. In addition, the recombinant enzyme has a remarkable fibrin(ogen)olytic activity. This indicates that the enzyme has a potential for application in cardiovascular diseases, besides its use in tanning as a dehairing agent.     

Manipulations of AMP metabolic genes increase growth rate and cold tolerance in Escherichia coli: implications for psychrophilic evolution

Diverse organisms have adapted to thrive at low temperatures (i.e., <20 °C, termed psychrophiles), colonizing the majority of earth's biosphere. In contrast with mesophiles (20-40 °C thermal range), all observed psychrophiles increase intracellular adenosine 5'-triphosphate concentrations as temperatures decline; this phenomenon has been described as an important compensatory mechanism to deal with decreases in thermal energy and molecular motion. We considered purine metabolic pathways in class gammaproteobacteria (n = 115) to investigate metabolic and evolutionary bases of this process. A survey of the KEGG database indicated that psychrophilic purine metabolic pathways tend to be enriched with de novo adenosine 5'-monophosphate (AMP) synthetic enzymes, whereas mesophiles tend to be enriched with AMP degradative enzymes. Function of the observed psychrophilic pathway structure was tested by engineering the mesophilic gammaproteobacterium Escherichia coli to reflect psychrophilic purine metabolism, specifically by expressing adenylosuccinate synthetase (purA) from the psychrophilic gammaproteobacterium, Psychrobacter cryohalolentis, in an AMP nucleosidase (amn)-deficient background. Modified E. coli was capable of growing up to ～70% faster at low temperatures and became up to ～10-fold more cold tolerant relative to wild type. These findings highlight potentially important transitional steps in psychrophilic evolution.     

Structural and functional adaptations to extreme temperatures in psychrophilic,mesophilic, and thermophilic DNA ligases

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.     

Psychrophilic and psychrotrophic fungi: a comprehensive review

This article reviews the comparative diversity of psychrophilic and psychrotrophic fungi, their adaptability mechanisms for survival and potential applications in biotechnology and pharmaceuticals. Fungi are able to grow and survive at low temperature and exist widely in polar and non-polar habitats. These cold regions are known for very low temperature, high ultra violet-B radiation, frequent freeze and thaw cycles and low water and nutrient availability. Most of the fungi adapt to such harsh conditions by evolving various strategies in their metabolism and physiology. Psychrophilic and psychrotrophic fungi are of importance in biotechnological and pharmaceutical fields due to their diverse characteristics developed or evolved due to their adaptation and survival in extreme environments, like; production of cold-active enzymes, pharmaceutical or bioactive metabolites and exo-polysaccharides, have potential for bioremediation and can also be used as biofertilizer.     

Cold active microbial lipases: some hot issues and recent developments

Lipases are glycerol ester hydrolases that catalyze the hydrolysis of triglycerides to free fatty acids and glycerol. Lipases catalyze esterification, interesterification, acidolysis, alcoholysis and aminolysis in addition to the hydrolytic activity on triglycerides. The temperature stability of lipases has regarded as the most important characteristic for use in industry. Psychrophilic lipases have lately attracted attention because of their increasing use in the organic synthesis of chiral intermediates due to their low optimum temperature and high activity at very low temperatures, which are favorable properties for the production of relatively frail compounds. In addition, these enzymes have an advantage under low water conditions due to their inherent greater flexibility, wherein the activity of mesophilic and thermophilic enzymes are severely impaired by an excess of rigidity. Cold-adapted microorganisms are potential source of cold-active lipases and they have been isolated from cold regions and studied. Compared to other lipases, relatively smaller numbers of cold active bacterial lipases were well studied. Lipases isolated from different sources have a wide range of properties depending on their sources with respect to positional specificity, fatty acid specificity, thermostability, pH optimum, etc. Use of industrial enzymes allows the technologist to develop processes that closely approach the gentle, efficient processes in nature. Some of these processes using cold active lipase from C. antarctica have been patented by pharmaceutical, chemical and food industries. Cold active lipases cover a broad spectrum of biotechnological applications like additives in detergents, additives in food industries, environmental bioremediations, biotransformation, molecular biology applications and heterologous gene expression in psychrophilic hosts to prevent formation of inclusion bodies. Cold active enzymes from psychrotrophic microorganisms showing high catalytic activity at low temperatures can be highly expressed in such recombinant strains. Thus, cold active lipases are today the enzymes of choice for organic chemists, pharmacists, biophysicists, biochemical and process engineers, biotechnologists, microbiologists and biochemists.     

Structural insights into cold inactivation of tryptophanase and cold adaptation of subtilisin S41

A wide variety of enzymes can undergo a reversible loss of activity at low temperature, a process that is termed cold inactivation. This phenomenon is found in oligomeric enzymes such as tryptophanase (Trpase) and other pyridoxal phosphate dependent enzymes. On the other hand, cold-adapted, or psychrophilic enzymes, isolated from organisms able to thrive in permanently cold environments, have optimal activity at low temperature, which is associated with low thermal stability. Since cold inactivation may be considered "contradictory" to cold adaptation, we have looked into the amino acid sequences and the crystal structures of two families of enzymes, subtilisin and tryptophanase. Two cold adapted subtilisins, S41 and subtilisin-like protease from Vibrio, were compared to a mesophilic and a thermophilic subtilisins, as well as to four PLP-dependent enzymes in order to understand the specific surface residues, specific interactions, or any other molecular features that may be responsible for the differences in their tolerance to cold temperatures. The comparison between the psychrophilic and the mesophilic subtilisins revealed that the cold adapted subtilisins have a high content of acidic residues mainly found on their surface, making it charged. The analysis of the Trpases showed that they have a high content of hydrophobic residues on their surface. Thus, we suggest that the negatively charged residues on the surface of the subtilisins may be responsible for their cold adaptation, whereas the hydrophobic residues on the surface of monomeric Trpase molecules are responsible for the tetrameric assembly, and may account for their cold inactivation and dissociation.     

Applications of deep eutectic solvents in biotechnology and bioengineering—Promises and challenges

Deep eutectic solvents (DESs) have been touted recently as potential alternatives to ionic liquids (ILs). Although they possess core characteristics that are similar to those of ILs (e.g., low volatility, non-flammability, low melting points, low vapor pressure, dipolar nature, chemical and thermal stability, high solubility, and tuneability), DESs are superior in terms of the availability of raw materials, the ease of storage and synthesis, and the low cost of their starting materials. As such, they have become the subject of intensive research in various sectors, notably the chemical, electrochemical, and biological sectors. To date, the applications of DESs have shown great promise, especially in the medical and biotechnological fields. In spite of these various achievements, the safety concern for these mixtures must be sufficiently addressed. Indeed, in order to exploit the vast array of opportunities that DESs offer to the biological industry, first, they must be established as safe mixtures. Hence, the biotechnological applications of DESs only can be implemented if they are proven to have negligible or low toxicity profiles. This review is the first of its kind, and it discusses two current aspects of DES-based research. First, it describes the properties of these mixtures with ample focus on their toxicity profiles. Second, it provides an overview of the breakthroughs that have occurred and the foreseeable prospects of the use of DESs in various biotechnological and biological applications.