A perspective on cold enzymes: current knowledge and frequently asked questions.

Studies on psychrophilic enzymes to determine the structural features important for cold-activity have attracted increased attention in the last few years. This enhanced interest is due to the attractive properties of such proteins, i.e. a high specific activity and a low thermal stability, and thus, these enzymes constitute a tremendous potential for fundamental research and biotechnological applications. This review examines the impact of low temperatures on life, the diversity of adaptation to counteract these effects and gives an overview of the features proposed to account for low thermal stability and cold-activity, following the chronological order of the catalytic cycle phases. Moreover, we present an overview of recent techniques used in the analysis of the flexibility of a protein structure which is an important concept in cold-adaptation; an overview of biotechnological potential of psychrophilic enzymes and finally, a few frequently asked questions about cold-adaptation and their possible answers.     

Functional motions of Candida antarctica lipase B: a survey through open-close conformations

Candida antarctica lipase B (CALB) belongs to psychrophilic lipases which hydrolyze carboxyl ester bonds at low temperatures. There have been some features reported about cold-activity of the enzyme through experimental methods, whereas there is no detailed information on its mechanism of action at molecular level. Herein, a comparative molecular dynamics simulation and essential dynamics analysis have been carried out at three temperatures (5, 35 and 50 °C) to trace the dominant factors in the psychrophilic properties of CALB under cold condition. The results clearly describe the effect of temperature on CALB with meaningful differences in the flexibility of the lid region (α5 helix), covering residues 141-147. Open- closed conformations have been obtained from different sets of long-term simulations (60 ns) at 5 °C gave two reproducible distinct forms of CALB. The starting open conformation became closed immediately at 35 and 50 °C during 60 ns of simulation, while a sequential open-closed form was observed at 5 °C. These structural alterations were resulted from α5 helical movements, where the closed conformation of active site cleft was formed by displacement of both helix and its side chains. Analysis of normal mode showed concerted motions that are involved in the movement of both α5 and α10 helices. It is suggested that the functional motions needed for lypolytic activity of CALB is constructed from short-range movement of α5, accompanied by long-range movement of the domains connected to the lid region.     

Activity–stability relationships revisited in blue oxidases catalyzing electron transfer at extreme temperatures

Cuproxidases are a subset of the blue multicopper oxidases that catalyze the oxidation of toxic Cu(I) ions into less harmful Cu(II) in the bacterial periplasm. Cuproxidases from psychrophilic, mesophilic, and thermophilic bacteria display the canonical features of temperature adaptation, such as increases in structural stability and apparent optimal temperature for activity with environmental temperature as well as increases in the binding affinity for catalytic and substrate copper ions. In contrast, the oxidative activities at 25 °C for both the psychrophilic and thermophilic enzymes are similar, suggesting that the nearly temperature-independent electron transfer rate does not require peculiar adjustments. Furthermore, the structural flexibilities of both the psychrophilic and thermophilic enzymes are also similar, indicating that the firm and precise bindings of the four catalytic copper ions are essential for the oxidase function. These results show that the requirements for enzymatic electron transfer, in the absence of the selective pressure of temperature on electron transfer rates, produce a specific adaptive pattern, which is distinct from that observed in enzymes possessing a well-defined active site and relying on conformational changes such as for the induced fit mechanism.     

Unscrambling thermal stability and temperature adaptation in evolved variants of a cold-active lipase

Directed evolution by error-prone PCR was applied to stabilize the cold-active lipase from Pseudomonas fragi (PFL). PFL displays high activity at 10 degrees C, but it is highly unstable even at moderate temperatures. After two rounds of evolution, a variant was generated with a 5-fold increase in half-life at 42 degrees C and a shift of 10 degrees C in the temperature optimum, nevertheless retaining cold-activity. The evolved lipase displayed specific activity higher than the wild type enzyme in the temperature range 29-42 degrees C. Biophysical measurements did not indicate any obvious difference between the improved variant and the wild type enzyme in terms of loss of secondary structure upon heat treatment, nor a shift in the apparent melting temperature.     

Bacterial Adaptation to Low Temperature: Implications for Cold-Inducible Genes

Escherichia coli and later found to be a cold-shock response common to many bacterial species. CspA of 7.4 kD, a major cold-shock protein in E. coli, has been shown to share structural similarity with a class of eukaryotic Y box proteins which have RNA-binding domains. Transient synthesis of CspA upon cold shock is mediated by increased stabilization of the mRNA at low temperatures. The proposed role of some cold-shock proteins including CspA in the bacterial adaptation to low temperatures is to function as a RNA chaperone in the regulation of translation. Some enzymes of psychrotrophic or psychrophilic bacteria exhibit unique features of a cold-adapted enzyme, high catalytic activity at a low temperature and rapid inactivation at a moderate temperature. A monomeric isocitrate dehydrogenase isozyme (IDH-II) of a psychrophilic bacterium, Vibrio sp. strain ABE-1, is a typical cold-adapted enzyme. In addition, this enzyme is induced at low temperatures. Low temperature-dependent expression of icdll encoding IDH-II is controlled by two different cis-elements located at the untranslated upstream region of the gene, one is a silencer and the other is essential for the low temperature response. The physiological role of IDH-II is evaluated by transforming E. coli with icdll. The growth rate of the E. coli transformants at low temperatures is dependent on the level of expressed IDH-II activity. Received 11 January 1999/ Accepted in revised form 6 April 1999     

Analysis of pH-dependent elements in proteins: geometry and properties of pairs of hydrogen-bonded carboxylic acid side-chains

A rather frequent but so far little discussed observation is that pairs of carboxylic acid side-chains in proteins can share a proton in a hydrogen bond. In the present article, quantum chemical calculations of simple model systems for carboxyl-carboxylate interactions are compared with structural observations from proteins. A detailed structural analysis of the proteins deposited in the PDB revealed that, in a subset of proteins sharing less than 90% sequence identity, 19% (314) contain at least one pair of carboxylic acids with their side-chain oxygen atoms within hydrogen-bonding distance. As the distance between those interacting oxygen atoms is frequently very short ( approximately 2.55 A), many of these carboxylic acids are suggested to share a proton in a strong hydrogen bond. When situated in an appropriate structural environment (low dielectric constant), some might even form a low barrier hydrogen bond. The quantum chemical studies show that the most frequent geometric features of carboxyl-carboxylate pairs found in proteins, and no or symmetric ligation, are also the most stable arrangements at low dielectric constants, and they also suggest at medium and low pH a higher stability than for isosteric amide-carboxylate pairs. The presence of these pairs in 119 different enzymes found in the BRENDA database is set in relation to their properties and functions. This analysis shows that pH optima of enzymes with carboxyl-carboxylate pairs are shifted to lower than average values, whereas temperature optima seem to be increased. The described structural principles can be used as guidelines for rational protein design (e.g., in order to improve pH or temperature stability).     

Insights into the role of reactive sulfhydryl groups of Carbonic Anhydrase III and VII during oxidative damage

Carbonic anhydrases (CAs) III and VII are two cytosolic isoforms of the α-CA family which catalyze the physiological reaction of carbon dioxide hydration to bicarbonate and proton. Despite these two enzymes share a 49% sequence identity and present a very similar three-dimensional structure, they show profound differences when comparing the specific activity for CO₂ hydration reaction, with CA VII being much more active than CA III. Recently, CA III and CA VII have been proposed to play a new role as scavenger enzymes in cells where oxidative damage occurs. Here, we will examine functional and structural features of these two isoforms giving insights into their newly proposed protective role against oxidative stress.     

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.     

Probing the S1 specificity pocket of the aminopeptidases that generate antigenic peptides

ERAP1 (endoplasmic reticulum aminopeptidase 1), ERAP2 and IRAP (insulin-regulated aminopeptidase) are three homologous enzymes that play critical roles in the generation of antigenic peptides. These aminopeptidases excise amino acids from N-terminally extended precursors of antigenic peptides in order to generate the correct length epitopes for binding on to MHC class I molecules. The specificity of these peptidases can affect antigenic peptide selection, but has not yet been investigated in detail. In the present study we utilized a collection of 82 fluorigenic substrates to define a detailed selectivity profile for each of the three enzymes and to probe structural and functional features of the S1 (primary specificity) pocket. Molecular modelling of the three S1 pockets reveals substrate-enzyme interactions that are critical determinants for specificity. The substrate selectivity profiles suggest that IRAP largely combines the S1 specificity of ERAP1 and ERAP2, consistent with its proposed biological function. IRAP, however, does not achieve this dual specificity by simply combining structural features of ERAP1 and ERAP2, but rather by an unique amino acid change at position 541. The results of the present study provide insights on antigenic peptide selection and may prove valuable in designing selective inhibitors or activity markers for this class of enzymes.     

Homologous yeast lipases/acyltransferases exhibit remarkable cold-active properties

Lipases/acyltransferases catalyse acyltransfer to various nucleophiles preferentially to hydrolysis even in aqueous media with high thermodynamic activity of water (a _w >0.9). Characterization of hydrolysis and acyltransfer activities in a large range of temperature (5 to 80 °C) of secreted recombinant homologous lipases of the Pseudozyma antarctica lipase A superfamily (CaLA) expressed in Pichia pastoris, enlighten the exceptional cold-activity of two remarkable lipases/acyltransferases: CpLIP2 from Candida parapsilosis and CtroL4 from Candida tropicalis. The activation energy of the reactions catalysed by CpLIP2 and CtroL4 was 18–23 kJ mol^?1 for hydrolysis and less than 15 kJ mol^?1 for transesterification between 5 and 35 °C, while it was respectively 43 and 47 kJ mol^?1 with the thermostable CaLA. A remarkable consequence is the high rate of the reactions catalysed by CpLIP2 and CtroL4 at very low temperatures, with CpLIP2 displaying at 5 °C 65 % of its alcoholysis activity and 45 % of its hydrolysis activity at 30 °C. These results suggest that, within the CaLA superfamily and its homologous subgroups, common structural determinants might allow both acyltransfer and cold-active properties. Such biocatalysts are of great interest for the efficient synthesis or functionalization of temperature-sensitive lipid derivatives, or more generally to lessen the environmental impact of biocatalytic processes.     

Flexibility and enzymatic cold-adaptation: a comparative molecular dynamics investigation of the elastase family

Molecular dynamics simulations of representative mesophilic and psycrophilic elastases have been carried out at different temperatures to explore the molecular basis of cold adaptation inside a specific enzymatic family. The molecular dynamics trajectories have been compared and analyzed in terms of secondary structure, molecular flexibility, intramolecular and protein-solvent interactions, unravelling molecular features relevant to rationalize the efficient catalytic activity of psychrophilic elastases at low temperature. The comparative molecular dynamics investigation reveals that modulation of the number of protein-solvent interactions is not the evolutionary strategy followed by the psycrophilic elastase to enhance catalytic activity at low temperature. In addition, flexibility and solvent accessibility of the residues forming the catalytic triad and the specificity pocket are comparable in the cold- and warm-adapted enzymes. Instead, loop regions with different amino acid composition in the two enzymes, and clustered around the active site or the specificity pocket, are characterized by enhanced flexibility in the cold-adapted enzyme. Remarkably, the psycrophilic elastase is characterized by reduced flexibility, when compared to the mesophilic counterpart, in some scattered regions distant from the functional sites, in agreement with hypothesis suggesting that local rigidity in regions far from functional sites can be beneficial for the catalytic activity of psychrophilic enzymes.     

Thermostable Artificial Enzyme Isolated by In Vitro Selection

Artificial enzymes hold the potential to catalyze valuable reactions not observed in nature. One approach to build artificial enzymes introduces mutations into an existing protein scaffold to enable a new catalytic activity. This process commonly results in a simultaneous reduction of protein stability as an undesired side effect. While protein stability can be increased through techniques like directed evolution, care needs to be taken that added stability, conversely, does not sacrifice the desired activity of the enzyme. Ideally, enzymatic activity and protein stability are engineered simultaneously to ensure that stable enzymes with the desired catalytic properties are isolated. Here, we present the use of the in vitro selection technique mRNA display to isolate enzymes with improved stability and activity in a single step. Starting with a library of artificial RNA ligase enzymes that were previously isolated at ambient temperature and were therefore mostly mesophilic, we selected for thermostable active enzyme variants by performing the selection step at 65°C. The most efficient enzyme, ligase 10C, was not only active at 65°C, but was also an order of magnitude more active at room temperature compared to related enzymes previously isolated at ambient temperature. Concurrently, the melting temperature of ligase 10C increased by 35 degrees compared to these related enzymes. While low stability and solubility of the previously selected enzymes prevented a structural characterization, the improved properties of the heat-stable ligase 10C finally allowed us to solve the three-dimensional structure by NMR. This artificial enzyme adopted an entirely novel fold that has not been seen in nature, which was published elsewhere. These results highlight the versatility of the in vitro selection technique mRNA display as a powerful method for the isolation of thermostable novel enzymes.     

Structure and function of Rubisco

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the major enzyme assimilating CO(2) into the biosphere. At the same time Rubisco is an extremely inefficient catalyst and its carboxylase activity is compromised by an opposing oxygenase activity involving atmospheric O(2). The shortcomings of Rubisco have implications for crop yield, nitrogen and water usage, and for the global carbon cycle. Numerous high-resolution crystal structures of different forms of Rubisco are now available, including structures of mutant enzymes. This review uses the information provided in these structures in a structure-based sequence alignment and discusses Rubisco function in the context of structural variations at all levels--amino acid sequence, fold, tertiary and quaternary structure--with an evolutionary perspective and an emphasis on the structural features of the enzyme that may determine its function as a carboxylase.     

温度对嗜冷酵母糖代谢途径某些关键酶的活性效应

对嗜冷酵母Y18和酿酒酵母细胞中EMP途径和TCA循环中一些关键酶的温度特性进行了比较研究。Y18细胞中,1,6二磷酸果糖醛缩酶、琥珀酸脱氢酶和己糖激酶对温度很敏感,符合Feller提出的冷活性的概念属于冷活性酶类。柠檬酸合成酶的温度特性类似于中温酶。Α酮戊二酸脱氢酶存在不同温度特性的同功酶。通过对嗜冷酵母和中温酶母细胞中琥珀酸脱氢酶的Km值进行比较,结果显示嗜冷酵母琥珀酸脱氢酶在20℃具有较低的Km值。另外还对嗜冷菌细胞中代谢酶类的一些特点进行了讨论。     

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.     

Job contenders: roles of the β‐barrel assembly machinery and the translocation and assembly module in autotransporter secretion

Billions of years of evolution have yielded today's complex metabolic networks driven by efficient and highly specialized enzymes. In contrast, the metabolism of the earliest cellular life forms was likely much simpler with only a few enzymes of comparatively low activity. It has been speculated that these early enzymes had low specificities and in turn were able to perform multiple functions. In this issue of Molecular Microbiology, Ferla et al. describe examples of enzymes that catalyze chemically distinct reactions while using the same active site. Most importantly, the authors demonstrated that the comparatively weak activities of these multifunctional enzymes are each physiologically relevant. These findings contrast with simply promiscuous enzyme activities, which have been described numerous times but are not physiologically relevant. Ferla et al. elegantly combined initial bioinformatics searches for enzyme candidates with sound kinetic measurements, evolutionary considerations and even structural discussions. The phenomenon of multifunctionality appears to be a mechanism for bacteria with reduced genomes to compensate for their lack of certain enzymes. In the broader context of evolution, these organisms could be considered living model systems to study features of long‐extinct early cellular life.     

Architecture of the catalytic HPN motif is conserved in all E2 conjugating enzymes

E2 conjugating enzymes are the central enzymes in the ubiquitination pathway and are responsible for the transfer of ubiquitin and ubiquitin-like proteins on to target substrates. The secondary structural elements of the catalytic domain of these enzymes is highly conserved, including the sequence conservation of a three-residue HPN (His-Pro-Asn) motif located upstream of the active-site cysteine residue used for ubiquitin conjugation. Despite the vast structural knowledge of E2 enzymes, the catalytic mechanism of these enzymes remains poorly understood, in large part due to variation in the arrangements of the residues in the HPN motif in existing E2 structures. In the present study, we used the E2 enzyme HIP2 to probe the structures of the HPN motif in several other E2 enzymes. A combination of chemical-shift analysis, determination of the histidine protonation states and amide temperature coefficients were used to determine the orientation of the histidine ring and hydrogen-bonding arrangements within the HPN motif. Unlike many three-dimensional structures, we found that a conserved hydrogen bond between the histidine imidazole ring and the asparagine backbone amide proton, a common histidine protonation state, and a common histidine orientation exists for all E2 enzymes examined. These results indicate that the histidine within the HPN motif is orientated to structurally stabilize a tight turn motif in all E2 enzymes and is not orientated to interact with the asparagine side chain as proposed in some mechanisms. These results suggest that a common catalysis mechanism probably exists for all E2 conjugating enzymes to facilitate ubiquitin transfer.     

Effect of pasteurization on the activity of proteinases in salmon roe

We studied the dependence of activity and stability of proteolytic enzymes in salmon roe on pH and temperature. The activity of proteolytic enzymes in roe was primarily determined by proteinases. These enzymes were active at acid pH and had an optimum of 3.6. A study of subclasses of proteolytic enzymes in salmon roe and the published data suggest that the activity of proteinases may be related to the presence of aspartyl proteinases (cathepsin D). Serine proteinases and metalloenzymes were not found in roe. The activity of cysteine proteinases was low. The proposed conditions of pasteurization favored the complete inactivation of salmon roe at pH 6.0-6.4.     

Effect of Pasteurization on the Activity of Proteinases in Salmon Roe

We studied the dependence of activity and stability of proteolytic enzymes in salmon roe on pH and temperature. The activity of proteolytic enzymes in roe was primarily determined by proteinases. These enzymes were active at acid pH and had an optimum of 3.6. A study of subclasses of proteolytic enzymes in salmon roe and the published data suggest that the activity of proteinases may be related to the presence of aspartyl proteinases (cathepsin D). Serine proteinases and metalloenzymes were not found in roe. The activity of cysteine proteinases was low. The proposed conditions of pasteurization favored the complete inactivation of salmon roe at pH 6.0–6.4.     

Molecular Structural Basis for the Cold Adaptedness of the Psychrophilic β-Glucosidase BglU in Micrococcus antarcticus

Psychrophilic enzymes play crucial roles in cold adaptation of microbes and provide useful models for studies of protein evolution, folding, and dynamic properties. We examined the crystal structure (2.2-Å resolution) of the psychrophilic β-glucosidase BglU, a member of the glycosyl hydrolase 1 (GH1) enzyme family found in the cold-adapted bacterium Micrococcus antarcticus. Structural comparison and sequence alignment between BglU and its mesophilic and thermophilic counterpart enzymes (BglB and GlyTn, respectively) revealed two notable features distinct to BglU: (i) a unique long-loop L3 (35 versus 7 amino acids in others) involved in substrate binding and (ii) a unique amino acid, His299 (Tyr in others), involved in the stabilization of an ordered water molecule chain. Shortening of loop L3 to 25 amino acids reduced low-temperature catalytic activity, substrate-binding ability, the optimal temperature, and the melting temperature (T_m). Mutation of His299 to Tyr increased the optimal temperature, the T_m, and the catalytic activity. Conversely, mutation of Tyr301 to His in BglB caused a reduction in catalytic activity, thermostability, and the optimal temperature (45 to 35°C). Loop L3 shortening and H299Y substitution jointly restored enzyme activity to the level of BglU, but at moderate temperatures. Our findings indicate that loop L3 controls the level of catalytic activity at low temperatures, residue His299 is responsible for thermolability (particularly heat lability of the active center), and long-loop L3 and His299 are jointly responsible for the psychrophilic properties. The described structural basis for the cold adaptedness of BglU will be helpful for structure-based engineering of new cold-adapted enzymes and for the production of mutants useful in a variety of industrial processes at different temperatures.