Stability and Stabilization of Enzymes from Mesophilic and Thermophilic Organisms in Respect to Their Use in FIA-Systems for the Determination of L-Lactate and Acetate

The stabilization effect of ‘bilayer encagement’ on enzymes from mesophilic organisms and their ‘thermophilic’ counterparts was compared. Lactate dehydrogenases from pig heart and from a thermophilic bacterium (Clostridium thermohydrosulfuricum), respectively, showed stabilization factors of 4·5 (at 47°C) and 12·8 (at 70°C), respectively. For ‘thermophilic’ acetate kinase no stabilization effect of encagement was observed. Lactate dehydrogenase and acetate kinase from Clostridium thermohydrosulfuricum were immobilized to controlled porous glass and tested for their long-term stability. The ‘thermophilic’ enzymes showed by far a longer half-life as compared with the corresponding enzymes from pig heart and Escherichia coli, respectively, the half-life time of the flow injection system response with thermophilic lactate dehydrogenase at 35°C attaining 250 h (mesophilic enzyme 89 h), and with thermophilic acetate kinase 79 h (mesophilic enzyme 24 h). © 1997 SCI.     

Serine Hydroxymethyltransferase from the Cold Adapted Microorganism Psychromonas ingrahamii: A Low Temperature Active Enzyme with Broad Substrate Specificity

Serine hydroxymethyltransferase from the psychrophilic microorganism Psychromonas ingrahamii was expressed in Escherichia coli and purified as a His-tag fusion protein. The enzyme was characterized with respect to its spectroscopic, catalytic, and thermodynamic properties. The properties of the psychrophilic enzyme have been contrasted with the characteristics of the homologous counterpart from E. coli, which has been structurally and functionally characterized in depth and with which it shares 75% sequence identity. Spectroscopic measures confirmed that the psychrophilic enzyme displays structural properties almost identical to those of the mesophilic counterpart. At variance, the P. ingrahamii enzyme showed decreased thermostability and high specific activity at low temperature, both of which are typical features of cold adapted enzymes. Furthermore, it was a more efficient biocatalyst compared to E. coli serine hydroxymethyltransferase (SHMT) particularly for side reactions. Many β-hydroxy-α-amino acids are SHMT substrates and represent important compounds in the synthesis of pharmaceuticals, agrochemicals and food additives. Thanks to these attractive properties, this enzyme could have a significant potential for biotechnological applications.     

Kinetic and structural characterization of thermostabilized mutants of human carbonic anhydrase II

Carbonic anhydrases (CAs) are ubiquitous enzymes that catalyze the reversible hydration/dehydration of carbon dioxide/bicarbonate. As such, there is enormous industrial interest in using CA as a bio-catalyst for carbon sequestration and biofuel production. However, to ensure cost-effective use of the enzyme under harsh industrial conditions, studies were initiated to produce variants with enhanced thermostability while retaining high solubility and catalytic activity. Kinetic and structural studies were conducted to determine the structural and functional effects of these mutations. X-ray crystallography revealed that a gain in surface hydrogen bonding contributes to stability while retaining proper active site geometry and electrostatics to sustain catalytic efficiency. The kinetic profiles determined under a variety of conditions show that the surface mutations did not negatively impact the carbon dioxide hydration or proton transfer activity of the enzyme. Together these results show that it is possible to enhance the thermal stability of human carbonic anhydrase II by specific replacements of surface hydrophobic residues of the enzyme. In addition, combining these stabilizing mutations with strategic active site changes have resulted in thermostable mutants with desirable kinetic properties.     

Sequence and homology model of 3-isopropylmalate dehydrogenase from the psychrotrophic bacterium Vibrio sp. I5 suggest reasons for thermal instability

The leuB gene from the psychrotrophic strain Vibrio sp. I5 has been cloned and sequenced. The gene codes for 3-isopropylmalate dehydrogenase, a 360-residue, dimeric enzyme involved in the biosynthesis of leucine. Three recently solved homologous isopropylmalate dehydrogenase (IPMDH) crystal structures from thermophilic and mesophilic organisms have been used to build a homology model for the psychrotrophic IPMDH and to deduce the possible structural reasons for its decreased thermostability. According to our model the psychrotrophic IPMDH contains fewer stabilizing interactions than its mesophilic and thermophilic counterparts. Elements that have been identified as destabilizing in the comparison of the psychrotrophic, mesophilic and thermophilic IPMDHs are a smaller number of salt-bridges, a reduction in aromatic-aromatic interactions, fewer proline residues and longer surface loops. In addition, there are a number of substitutions of otherwise strictly conserved residues that can be linked to thermostability.      

Adaptation of class-13 alpha-amylases to diverse living conditions

There are currently 35 available nonredundant molecular structures of class-13 alpha-amylases (EC 3.2.1.1), mostly from microbial organisms living under a wide range of environmental conditions. One of the most recent additions has been the first alpha-amylase structure of a hyperthermophilic archaeon [Linden et al., J. Biol. Chem. 2003, 278, 9875-9884]. The structure has been used for comparative analyses with a representative set of three alpha-amylases from thermophilic, mesophilic and psychrophilic sources to identify molecular parameters for environmental adaptation. Our analysis supports generally observed trends such as an increase in structural compactness as well as an increase in salt bridges in order to cope with high-temperature conditions. The two representative thermophilic structures used in this comparative study have independently evolved di-metal centres--not present in the mesophilic and psychrophilic structures--in the vicinity of the active site. These observations may provide impetus for the design of alpha-amylases with improved molecular properties to enhance their utility in biotechnological processes.     

Adaptation of a thermophilic enzyme, 3-isopropylmalate dehydrogenase, to low temperatures

Random mutagenesis coupled with screening of the active enzymeat a low temperature was applied to isolate cold-adapted mutantsof a thermophilic enzyme. Four mutant enzymes with enhancedspecific activities (up to 4.1-fold at 40°C) at a moderatetemperature were isolated from randomly mutated Thermus thermophilus3-isopropylmalate dehydrogenase. Kinetic analysis revealed twotypes of cold-adapted mutants, i.e. k_cat-improved and K_m-improvedtypes. The k_cat-improved mutants showed less temperature-dependentcatalytic properties, resulting in improvement of k_cat (up to7.5-fold at 40°C) at lower temperatures with increased K_mvalues mainly for NAD. The K_m-improved enzyme showed higheraffinities toward the substrate and the coenzyme without significantchange in k_cat at the temperatures investigated (30–70°C).In k_cat-improved mutants, replacement of a residue was foundnear the binding pocket for the adenine portion of NAD. Twoof the mutants retained thermal stability indistinguishablefrom the wild-type enzyme. Extreme thermal stability of thethermophilic enzyme is not necessarily decreased to improvethe catalytic function at lower temperatures. The present strategyprovides a powerful tool for obtaining active mutant enzymesat lower temperatures. The results also indicate that it ispossible to obtain cold-adapted mutant enzymes with high thermalstability.     

Stability of aspartate aminotransferase from Sulfolobus solfataricus

Aspartate aminotransferase from Sulfolobus solfataricus (SsAspAT) is an extremely thermophilic and thermostable dimeric enzyme which retains its structure and reaches maximal activity at 100 degrees C. The structural stability of this protein was investigated by coupling isothermally and thermally induced denaturation studies to molecular modeling. Gel filtration analysis indicated that SsAspAT unfolds with an N2 reversible 2D mechanism. In the molecular model, a cluster of hydrophobic residues was shown at the interface between the subunits of SsAspAT and suggested this cluster as a structural feature stabilizing the enzyme quaternary structure. At 25 degrees C, SsAspAT is less resistant to guanidinium chloride-induced denaturation than the cytosolic aspartate aminotransferase from pig heart (cpAspAT), which was chosen as a mesophilic counterpart in the thermodynamic analysis since it shares with SsAspAT the two-state unfolding mechanism. Therefore, in the case of aspartate aminotransferases, thermal stability does not correlate with the stability against chemical denaturants. Isothermal denaturation curves at 25 degrees C and melting profiles recorded in the presence of guanidinium chloride showed that the delta G degrees (H2O) at 25 degrees C of SsAspAT exceeds that of cpAspAT by roughly 15 kJ/mol; the parameter delta n, related to the number of binding sites for the denaturant differentially exposed in unfolded and folded states, is higher for SsAspAT than for cpAspAT; and delta Cp is lower for the thermophilic enzyme than for the mesophilic one by 8 kJ/K.mol. These results are indicative of a less hydrophobic core for SsAspAT than cpAspAT. In agreement with this, the molecular model predicts that some charged side chains are buried in SsAspAT and interact to form an H-bond/ion-pair network.      

Stabilization of a metabolic enzyme by library selection in Thermus thermophilus

The anthranilate phosphoribosyl transferase from the hyperthermophilic archaeon Sulfolobus solfataricus (sAnPRT, encoded by strpD), which catalyzes the third step in tryptophan biosynthesis, is a thermostable homodimer with low enzymatic activity at room temperature. We have combined two mutations leading to the monomerization and two mutations leading to the activation of sAnPRT. The resulting “activated monomer” sAnPRT‐I36E‐M47D+D83G‐F149S, which is much more labile than wild‐type sAnPRT, was stabilized by a combination of random mutagenesis and metabolic library selection using the extremely thermophilic bacterium Thermus thermophilus as host. This approach led to the identification of five mutations that individually increased the thermal stability of sAnPRT‐I36E‐M47D+D83G‐F149S by 1 to 8 °C, and by 13 °C when combined. The beneficial exchanges were located in different parts of the protein structure, but none of them led to the “re‐dimerization” of the enzyme. We observed a negative correlation between thermal stability and catalytic activity of the mutants; this suggests that conformational flexibility is required for catalysis by sAnPRT.     

Improvement of thermal stability of Streptomyces cholesterol oxidase by random mutagenesis and a structural interpretation

Random mutagenesis was used to enhance the thermal stability of Streptomyces cholesterol oxidase. Four thermostable mutants were isolated and the following amino acid substitutions were identified: Ser103 to Thr (mutant S103T), Val121 to Ala (mutant V121A), Arg135 to His (mutant R135H) and Val145 to Glu (mutant V145E). The wild-type and mutant enzymes were purified and characterized. The properties of mutants S103T, V121A and R135H were similar to those of the wild type but they showed improved thermal stability. When the V145E mutation was introduced, the thermal stability of the enzyme was markedly increased and the optimum pH was desirably changed to encompass a broad range from acid to alkali. Analysis of multiple mutants constructed by site- directed mutagenesis showed that all the mutations except that of R135H had an additive influence on the other mutations. These mutational effects are discussed in terms of a three-dimensional structural model of the enzyme constructed on the basis of homology modelling.      

Homology modelling of two subtilisin-like proteases from the hyperthermophilic archaea Pyrococcus furiosus and Thermococcus stetteri

The hyperthermophilic archaeon Pyrococcus furiosus produces an extracellular, glycosylated hyperthermostable subtilisin-like serine protease, termed pyrolysin (Voorhorst,W.G.B., Eggen,R.I.L., Geerling,A.C.M., Platteeuw,C., Siezen,R.J. and de Vos,W.M. (1996) J. Biol. Chem., 271, 20426-20431). Based on the pyrolysin coding sequence, a pyrolysin-like gene fragment was cloned and characterized from the extreme thermophilic archaeon Thermococcus stetteri. Like pyrolysin, the deduced sequence of this serine protease, designated stetterlysin, contains a catalytic domain with high homology with other subtilases, allowing homology modelling starting from known crystal structures. Comparison of the predicted three-dimensional models of the catalytic domain of stetterlysin and pyrolysin with the crystal structure of subtilases from mesophilic and thermophilic origin, i.e. subtilisin BPN' and thermitase, and the homology model of subtilisin S41 from psychrophilic origin, led to the identification of features that could be related to protein stabilization. Higher thermostability was found to be correlated with an increased number of residues involved in pairs and networks of charge-charge and aromatic-aromatic interactions. These highly thermostable proteases have several extra surface loops and inserts with a relatively high frequency of aromatic residues and Asn residues. The latter are often present in putative N-glycosylation sites. Results from modelling of known substrates in the substrate- binding region support the broad substrate range and the autocatalytic activation previously suggested for pyrolysin.      

Directed evolution by accumulating tailored mutations: thermostabilization of lactate oxidase with less trade-off with catalytic activity

We assumed that adverse effects posed by introducing multiple mutations could be decomposed into those of each of the component mutations and that the risk could be reduced by the accumulation of mutations that were finely tuned for directed improvement of a specific property. We propose here a directed evolution strategy for improving a specific property with less effect on other ones. This strategy is composed of fine-tuning of mutations and their accumulation by our original mutation-assembling method. In this study, we selected lactate oxidase (LOX) as a model enzyme, because its directed evolution had showed a trade-off between thermostability and catalytic activity. Mutation profiling at each of the sites found by error-prone PCR revealed a strong inverse relationship between the two properties. Thermostable mutations with less effect on catalytic activity were selected at each site and accumulated with ideal combinations by our method. The resultant multiple mutants exhibited 5- to 10-fold superior catalytic activity and comparable thermostability with those created by accumulating thermostable mutations, which were not tuned for catalytic activity. This result demonstrates that the accumulation of fine-tuned mutations is an advantageous approach to reduce the risk of adverse effects posed by accumulating multiple mutations.     

Decreasing the stability and changing the substrate specificity of the Bacillus stearothermophilus alcohol dehydrogenase by single amino acid replacements

The gene encoding the alcohol dehydrogenase (adh-hT) from the thermophilic bacterium Bacillus stearothermophilus LLD-R strain has been overexpressed in Escherichia coli and the corresponding recombinant protein purified to homogeneity. Two putative structural determinants contributing to the higher stability of ADH-hT had been identified by comparison with the less thermostable ADH (ADH-T) from the less thermophilic B. stearothermophilus NCA 1503. In order to ascertain their role, mutations were designed to eliminate in ADH-hT a salt bridge at the N-terminus and a proline residue in the coenzyme binding domain replacing the amino acids located at the same positions in ADH-T. Three mutants--Glu11Lys, Pro242Ala, and Glu11Lys/Pro242Ala-- were expressed at high level and the proteins purified and characterized. In general, the mutations had little effect on the activity, indicating that they were not disruptive. The thermal resistance was changed displaying quite additive effects.      

Different roles of electrostatics in heat and in cold: adaptation by citrate synthase

Electrostatics plays a major role in heat adaptation by thermophilic proteins. Here we ask whether electrostatics similarly contributes to cold adaptation in psychrophilic proteins. We compare the sequences and structures of citrate synthases from the psychrophile Arthobacter Ds2-3R, from chicken, and from the hyperthermophile Pyrococcus furiosus. The three enzymes share similar packing, burial of nonpolar surface area, and main-chain hydrogen bonding. However, both psychrophilic and hyperthermophilic citrate synthases contain more charged residues, salt bridges, and salt-bridge networks than the mesophile. The electrostatic free-energy contributions toward protein stability by individual charged residues show greater variabilities in the psychrophilic citrate synthase than in the hyperthermophilic enzyme. The charged residues in the active-site regions of the psychrophile are more destabilizing than those in the active-site regions of the hyperthermophile. In the hyperthermophilic enzyme, salt bridges and their networks largely cluster in the active-site regions and at the dimer interface. In contrast, in the psychrophile, they are more dispersed throughout the structure. On average, salt bridges and their networks provide greater electrostatic stabilization to the thermophilic citrate synthase at 100 degrees C than to the psychrophilic enzyme at 0 degrees C. Electrostatics appears to play an important role in both heat and cold adaptation of citrate synthase. However, remarkably, the role may be different in the two types of enzyme: In the hyperthermophile, it may contribute to the integrity of both the protein dimer and the active site by possibly countering conformational disorder at high temperatures. On the other hand, in the psychrophile at low temperatures, electrostatics may contribute to enhance protein solvation and to ensure active-site flexibility.     

Optimization to Low Temperature Activity in Psychrophilic Enzymes

Psychrophiles, i.e., organisms thriving permanently at near-zero temperatures, synthesize cold-active enzymes to sustain their cell cycle. These enzymes are already used in many biotechnological applications requiring high activity at mild temperatures or fast heat-inactivation rate. Most psychrophilic enzymes optimize a high activity at low temperature at the expense of substrate affinity, therefore reducing the free energy barrier of the transition state. Furthermore, a weak temperature dependence of activity ensures moderate reduction of the catalytic activity in the cold. In these naturally evolved enzymes, the optimization to low temperature activity is reached via destabilization of the structures bearing the active site or by destabilization of the whole molecule. This involves a reduction in the number and strength of all types of weak interactions or the disappearance of stability factors, resulting in improved dynamics of active site residues in the cold. Considering the subtle structural adjustments required for low temperature activity, directed evolution appears to be the most suitable methodology to engineer cold activity in biological catalysts.     

Molecular Elucidation of a Urate Oxidase from Deinococcus radiodurans for Hyperuricemia and Gout Therapy

Urate oxidase initiates the uric acid degradation pathways and is extensively used for protein drug development for gout therapy and serum uric acid diagnosis. We first present the biochemical and structural elucidation of a urate oxidase from the extremophile microorganism Deinococcus radiodurans (DrUox). From enzyme characterization, DrUox showed optimal catalytic ability at 30 °C and pH 9.0 with high stability under physiological conditions. Only the Mg²⁺ ion moderately elevated its activity, which indicates the characteristic of the cofactor-free urate oxidase family. Of note, DrUox is thermostable in mesophilic conditions. It retains almost 100% activity when incubated at 25 °C and 37 °C for 24 h. In this study, we characterized a thermostable urate oxidase, DrUox with high catalytic efficiency and thermal stability, which strengthens its potential for medical applications.     

Structural adaptation of enzymes to low temperatures

A systematic comparative analysis of 21 psychrophilic enzymesbelonging to different structural families from prokaryoticand eukaryotic organisms is reported. The sequences of theseenzymes were multiply aligned to 427 homologous proteins frommesophiles and thermophiles. The net flux of amino acid exchangesfrom meso/thermophilic to psychrophilic enzymes was measured.To assign the observed preferred exchanges to different structuralenvironments, such as secondary structure, solvent accessibilityand subunit interfaces, homology modeling was utilized to predictthe secondary structure and accessibility of amino acid residuesfor the psychrophilic enzymes for which no experimental three-dimensionalstructure is available. Our results show a clear tendency forthe charged residues Arg and Glu to be replaced at exposed siteson     

Thermostabilization of firefly luciferase by in vivo directed evolution

Firefly luciferase is widely used in a number of areas of biotechnology and molecular biology. However, rapid inactivation of wild-type (WT) luciferases at elevated temperatures often hampers their application. A simple non-lethal in vivo screening scheme was used to identify thermostable mutants of luciferase in Escherichia coli colonies. This scheme allowed carrying out each cycle of mutagenesis in a rapid and efficient manner. Four rounds of directed evolution were conducted on a part of the gene coding for amino acid residues 130-390 of Luciola mingrelica luciferase. The resultant mutant designated 4TS had a half-life of 10 h at 42°C, which is 65-fold higher compared with the WT luciferase. Moreover, the mutant 4TS showed a 1.9-fold increase in specific activity, 5.7-fold reduction of K(m) for ATP and a higher-temperature optimum compared with the WT enzyme. 4TS contains eight mutations, four?of which are suggested to be mainly responsible for the enhancement of thermostability: R211L, A217V, E356K and S364C. Thus, directed evolution with non-lethal colony screening for in vivo bioluminescence activity proved to be an effective and efficient approach for increasing thermal stability of luciferase while retaining high catalytic activity.     

Subtilisin from psychrophilic antarctic bacteria: characterization and site-directed mutagenesis of residues possibly involved in the adaptation to cold

A subtilisin excreted by the Antarctic Bacillus TA39 has been purified to homogeneity and characterised. Two independent genes subt1 and subt2 are present but only subt1 is expressed significantly in the culture medium. The enzyme displays the usual characteristics of cold enzymes i.e. a high catalytic efficiency at low and moderate temperatures and an increased thermosensitivity originating from a 3D structure probably more flexible than its mesophilic counterpart. This is corroborated by the analysis of the computerized structure which shows a significant decrease in the number and strength of intramolecular weak bonds such as salt bridges and aromatic interactions. The affinity for calcium is also almost three orders of magnitude lower than that of mesophilic subtilisin and the interactions with the solvent are significantly higher thanks to a large increase in the number of Asp residues in the loops connecting secondary structures. The relation between flexibility and activity was investigated by site-directed mutagenesis tending mainly to increase the rigidity of the molecular edifice through the incorporation of additional salt bridge, disulfide bridge, aromatic interaction and by increasing the affinity of the enzyme for calcium. An important stabilization of the molecular structure was achieved through a modification of a calcium ligand T85D. The thermostability of the mutated product expressed in a mesophilic Bacillus reaches that of mesophilic subtilisin. Most important is the fact that this mutation further enhances the specific activity by a factor close to 2 when compared to the wild type enzyme so that the overall activity of the mutated cold enzyme is about 20 times higher than that of mesophilic subtilisin, illustrating the fact that thermostability is not systematically inversely related to specific activity. This opens new perspectives in the use of cold enzymes in biotechnology.      

Catalytic promiscuity in the alpha/beta-hydrolase superfamily: hydroxamic acid formation, C--C bond formation, ester and thioester hydrolysis in the C--C hydrolase family

The haloperoxidase family of alpha/beta-hydrolases contains enzymes of several different catalytic activities, including esterases, C--C hydrolases and cofactor-independent haloperoxidases (perhydrolases), but the molecular basis of this catalytic promiscuity is not fully understood. The C--C hydrolase enzyme MhpC from E. coli is shown to possess esterase and thioesterase activity, and the ability to activate hydroxylamine as a nucleophile to form hydroxamic acid products. The ratio of these activities was examined for nine site-directed mutant enzymes that contained mutations at nonessential residues in the enzyme active site. Higher levels of esterase and thioesterase activity were found in mutants Phe173Gly and Trp264Gly; this might be due to increased amounts of space in the active site. Higher levels of hydroxamic acid formation activity were found in mutant Asn109His-a mutation found in many haloperoxidase enzymes. Wild-type and mutant MhpC enzymes were also capable of C--C bond formation in organic solvents, and the highest activity was observed in nonpolar solvents. The results provide experimental support for the catalytic promiscuity shown in this family of enzymes, and indicate that differences in catalytic function can be introduced by point mutations.     

Contribution of engineered electrostatic interactions to the stability of cytosolic malate dehydrogenase

Protein engineering is a promising tool to obtain stable proteins.Comparison between homologous thermophilic and mesophilic enzymesfrom a given structural family can reveal structural featuresresponsible for the enhanced stability of thermophilic proteins.Structures from pig heart cytosolic and Thermus flavus malatedehydrogenases (cMDH, Tf MDH), two proteins showing a 55% sequencehomology, were compared with the aim of increasing cMDH stabilityusing features from the Thermus flavus enzyme. Three potentialsalt bridges from Tf MDH were selected on the basis of theirlocation in the protein (surface R176-D200, inter-subunit E57–K168and intrasubunit R149–E275) and implemented on cMDH usingsite-directed mutagenesis. Mutants containing E275 were notproduced in any detectable amount, which shows that the energypenalty of introducing a charge imbalance in a region that wasnot exposed to solvent was too unfavourable to allow properfolding of the protein. The salt bridge R149–E275, ifformed, would not enhance stability enough to overcome thiseffect. The remaining mutants were expressed and active andno differences from wild-type other than stability were found.Of the mutants assayed, Q57E/L168K led to a stability increaseof 0.4 kcal/mol, as determined by either guanidinium chloridedenaturalization or thermal inactivation experiments. This resultsin a 15°C shift in the optimal temperature, thus confirmingthat the inter-subunit salt bridge initially present in theT.flavus enzyme was formed in the cMDH structure and that theextra energy obtained is transformed into an increase in proteinstability. These results indicate that the use of structuralfeatures of thermophilic enzymes, revealed by a detailed comparisonof three-dimensional structures, is a valid strategy to improvethe stability of mesophilic malate dehydrogenases.