Relation between stability, dynamics and enzyme activity in 3-phosphoglycerate kinases from yeast and Thermus thermophilus.

3-Phosphoglycerate kinases from yeast and the extreme thermophilic bacterium Thermus thermophilus HB8 have been used as models for investigating the relationship between stability, dynamics and activity. It was found that while at a given temperature the thermophilic protein is more stable, its conformational dynamics as measured by the ability of acrylamide to quench the fluorescence of a buried tryptophan as well as its specific activity, are both lower than for the mesophilic protein. As the temperature is increased, the thermodynamic stability of the thermophilic protein approaches that of the mesophilic protein at its working temperature. Its conformational dynamics and specific activity however were both shown to increase, until at the physiologically operational temperature, they become similar to those of the mesophilic enzyme at its operational temperature. These results confirm the proposal that a direct relationship and balance holds between thermodynamic stability, dynamics and specific activity in globular proteins. They demonstrate also the constraining effect of increased stability upon conformational dynamics and enzyme activity.     

Fundamental and biotechnological applications of neutron scattering measurements for macromolecular dynamics

To explore macromolecular dynamics on the picosecond timescale, we used neutron spectroscopy. First, molecular dynamics were analyzed for the hyperthermophile malate dehydrogenase from Methanococcus jannaschii and a mesophilic homologue, the lactate dehydrogenase from Oryctolagus cunniculus muscle. Hyperthermophiles have elaborate molecular mechanisms of adaptation to extremely high temperature. Using a novel elastic neutron scattering approach that provides independent measurements of the global flexibility and of the structural resilience (rigidity), we have demonstrated that macromolecular dynamics represents one of these molecular mechanisms of thermoadaptation. The flexibilities were found to be similar for both enzymes at their optimal activity temperature and the resilience is higher for the hyperthermophilic protein. Secondly, macromolecular motions were examined in a native and immobilized dihydrofolate reductase (DHFR) from Escherichia coli. The immobilized mesophilic enzyme has increased stability and decreased activity, so that its properties are changed to resemble those of the thermophilic enzyme. Are these changes reflected in dynamical behavior? For this study, we performed quasielastic neutron scattering measurements to probe the protein motions. The residence time is 7.95 ps for the native DHFR and 20.36 ps for the immobilized DHFR. The average height of the potential barrier to local motions is therefore increased in the immobilized DHFR, with a difference in activation energy equal to 0.54 kcal/mol, which is, using the theoretical rate equation, of the same order than expected from calculation.     

Temperature adaptation of lactate dehydrogenase. Structural, functional and genetic aspects

Comparison of the primary structures of thermophilic, mesophilic and psychrophilic lactate dehydrogenase (LDH) reveals a multitude of temperature-related amino acid substitutions. In the substitutions amino acid residues occurring preferentially in thermophilic, mesophilic (psychrophilic) LDH were found. On this basis, amino acid residues could be classified in an order from typical thermophilic (thermostabilizing) to typical mesophilic (thermolabilizing, increasing dynamics of the enzyme molecule) residues. The temperature-dependent ratio between thermostabilizing and thermolabilizing amino acid residues forms the basis for the specific structural and functional properties of thermophilic or mesophilic LDH. It is interesting that there appears to be a relationship between this order from thermophilic to mesophilic amino acid residues and the type of bases coding for these individual residues in the translation step of protein biosynthesis. Temperature-related amino acid substitutions are based on temperature-related base substitutions. A possible mechanism of temperature adaptation of LDH through alternative selection of thermophilic and mesophilic amino acid residues at the level of tRNA (anticodon)-mRNA (codon) interactions is discussed. These temperature-adaptation processes are evolutionary events in which the evolution and structure of the genetic code are involved.     

Structural equilibrium fluctuations in mesophilic and thermophilic alpha-amylase

By comparing a mesophilic alpha-amylase with its thermophilic homolog, we investigated the relationship between thermal stability and internal equilibrium fluctuations. Fourier transform infrared spectroscopy monitoring hydrogen/deuterium (H/D) exchange kinetics and incoherent neutron scattering measuring picosecond dynamics were used to study dynamic features of the folded state at room temperature. Fairly similar rates of slowly exchanging amide protons indicate about the same free energy of stabilization DeltaG(stab) for both enzymes at room temperature. With respect to motions on shorter time scales, the thermophilic enzyme is characterized by an unexpected higher structural flexibility as compared to the mesophilic counterpart. In particular, the picosecond dynamics revealed a higher degree of conformational freedom for the thermophilic alpha-amylase. The mechanism proposed for increasing thermal stability in the present case is characterized by entropic stabilization and by flattening of the curvature of DeltaG(stab) as a function of temperature.     

A comparative molecular dynamics study of thermophilic and mesophilic ribonuclease HI enzymes

We studied a pair of homologous thermophilic and mesophilic ribonuclease HI enzymes by molecular dynamics simulations. Each protein was subjected to three 5 ns simulations in explicit water at both 310 K and 340 K. The thermophilic enzyme showed larger overall positional fluctuations at both temperatures, while only the mesophilic enzyme at the higher temperature showed significant instability. When the temperature is changed, the relative flexibility of different local segments on the two proteins changed differently. Principal component analysis showed that the simulations of the two proteins explored largely overlapping regions in the conformational space. However, at 340 K, the collective structure variations of the thermophilic protein are different from those of the mesophilic protein. Our results, although not in accordance with the view that hyperthermostability of proteins may originate from their conformational rigidity, are consistent with several recent experimental and simulation studies which showed that thermophilic proteins may be conformationally more flexible than their mesophilic counterparts. The decorrelation between conformational rigidity and hyperthermostability may be attributed to the temperature dependence and long range nature of electrostatic interactions that play more important roles in the structural stability of thermophilic proteins.     

A Comparative Molecular Dynamics Study of Thermophilic and Mesophilic Ribonuclease HI Enzymes

Abstract

We studied a pair of homologous thermophilic and mesophilic ribonuclease HI enzymes by molecular dynamics simulations. Each protein was subjected to three 5 ns simulations in explicit water at both 310 K and 340 K. The thermophilic enzyme showed larger overall positional fluctuations at both temperatures, while only the mesophilic enzyme at the higher temperature showed significant instability. When the temperature is changed, the relative flexibility of different local segments on the two proteins changed differently. Principal component analysis showed that the simulations of the two proteins explored largely overlapping regions in the conformational space. However, at 340 K, the collective structure variations of the thermophilic protein are different from those of the mesophilic protein. Our results, although not in accordance with the view that hyperthermostability of proteins may originate from their conformational rigidity, are consistent with several recent experimental and simulation studies which showed that thermophilic proteins may be conformationally more flexible than their mesophilic counterparts. The decorrelation between conformational rigidity and hyperthermostability may be attributed to the temperature dependence and long range nature of electrostatic interactions that play more important roles in the structural stability of thermophilic proteins.     

Adaptation to high temperatures through macromolecular dynamics by neutron scattering

Work on the relationship between hyperthermophile protein dynamics, stability and activity is reviewed. Neutron spectroscopy has been applied to measure and compare the macromolecular dynamics of various hyperthermophilic and mesophilic proteins, under different conditions. First, molecular dynamics have been analyzed for the hyperthermophile malate dehydrogenase from Methanococcus jannaschii and a mesophilic homologue, the lactate dehydrogenase from Oryctolagus cunniculus (rabbit) muscle. The neutron scattering approach has provided independent measurements of the global flexibility and structural resilience of each protein, and it has been demonstrated that macromolecular dynamics represents one of the molecular mechanisms of thermoadaptation. The resilience was found to be higher for the hyperthermophilic protein, thus ensuring similar flexibilities in both enzymes at their optimal activity temperature. Second, the neutron method has been developed to quantify the average macromolecular flexibility and resilience within the natural crowded environment of the cell, and mean macromolecular motions have been measured in vivo in psychrophile, mesophile, thermophile and hyperthermophile bacteria. The macromolecular resilience in bacteria was found to increase with adaptation to high temperatures, whereas flexibility was maintained within narrow limits, independent of physiological temperature for all cells in their active state. Third, macromolecular motions have been measured in free and immobilized dihydrofolate reductase from Escherichia coli. The immobilized mesophilic enzyme has increased stability and decreased activity, so that its properties are changed to resemble those of a thermophilic enzyme. Quasi-elastic neutron scattering measurements have also been performed to probe the protein motions. Compared to the free enzyme, the average height of the activation free energy barrier to local motions was found to be increased by 0.54 kcal.mol(-1) in the immobilized dihydrofolate reductase, a value that is of the same order as expected from the theoretical rate equation.     

Comparing Residue Clusters from Thermophilic and Mesophilic Enzymes Reveals Adaptive Mechanisms

Understanding how proteins adapt to function at high temperatures is important for deciphering the energetics that dictate protein stability and folding. While multiple principles important for thermostability have been identified, we lack a unified understanding of how internal protein structural and chemical environment determine qualitative or quantitative impact of evolutionary mutations. In this work we compare equivalent clusters of spatially neighboring residues between paired thermophilic and mesophilic homologues to evaluate adaptations under the selective pressure of high temperature. We find the residue clusters in thermophilic enzymes generally display improved atomic packing compared to mesophilic enzymes, in agreement with previous research. Unlike residue clusters from mesophilic enzymes, however, thermophilic residue clusters do not have significant cavities. In addition, anchor residues found in many clusters are highly conserved with respect to atomic packing between both thermophilic and mesophilic enzymes. Thus the improvements in atomic packing observed in thermophilic homologues are not derived from these anchor residues but from neighboring positions, which may serve to expand optimized protein core regions.     

Atomic mean-square displacements in proteins by molecular dynamics: a case for analysis of variance

Information on protein internal motions is usually obtained through the analysis of atomic mean-square displacements, which are a measure of variability of the atomic positions distribution functions. We report a statistical approach to analyze molecular dynamics data on these displacements that is based on probability distribution functions. Using a technique inspired by the analysis of variance, we compute unbiased, reliable mean-square displacements of the atoms and analyze them statistically. We applied this procedure to characterize protein thermostability by comparing the results for a thermophilic enzyme and a mesophilic homolog. In agreement with previous experimental observations, our analysis suggests that the proteins surface regions can play a role in the different thermal behavior.     

Role of loop dynamics in thermal stability of mesophilic and thermophilic adenylosuccinate synthetase: a molecular dynamics and normal mode analysis study

Enzymes from thermophiles are poorly active at temperatures at which their mesophilic homologs exhibit high activity and attain corresponding active states at high temperatures. In this study, comparative molecular dynamics (MD) simulations, supplemented by normal mode analysis, have been performed on an enzyme Adenylosuccinate synthetase (AdSS) from E. coli (mesophilic) and P. horikoshii (thermophilic) systems to understand the effects of loop dynamics on thermal stability of AdSS. In mesophilic AdSS, both ligand binding and catalysis are facilitated through the coordinated movement of five loops on the protein. The simulation results suggest that thermophilic P. horikoshii preserves structure and catalytic function at high temperatures by using the movement of only a subset of loops (two out of five) for ligand binding and catalysis unlike its mesophilic counterpart in E. coli. The pre-arrangement of the catalytic residues in P. horikoshii is well-preserved and salt bridges remain stable at high temperature (363K). The simulations suggest a general mechanism (including pre-arrangement of catalytic residues, increased polar residue content, stable salt bridges, increased rigidity, and fewer loop movements) used by thermophilic enzymes to preserve structure and be catalytically active at elevated temperatures.     

Crowding induces differences in the diffusion of thermophilic and mesophilic proteins: a new look at neutron scattering results

The dynamical basis underlying the increased thermal stability of thermophilic proteins remains uncertain. Here, we challenge the new paradigm established by neutron scattering experiments in solution, in which the adaptation of thermophilic proteins to high temperatures lies in the lower sensitivity of their flexibility to temperature changes. By means of a combination of molecular dynamics and Brownian dynamics simulations, we report a reinterpretation of those experiments and show evidence that under crowding conditions, such as in vivo, thermophilic and homolog mesophilic proteins have diffusional properties with different thermal behavior.     

Differences in the catalytic mechanisms of mesophilic and thermophilic indole-3-glycerol phosphate synthase enzymes at their adaptive temperatures

Thermophilic enzymes tend to be less catalytically-active at lower temperatures relative to their mesophilic counterparts, despite having very similar crystal structures. An often cited hypothesis for this general observation is that thermostable enzymes have evolved a more rigid tertiary structure in order to cope with their more extreme, natural environment, but they are also less flexible at lower temperatures, leading to their lower catalytic activity under mesophilic conditions. An alternative hypothesis, however, is that complementary thermophilic-mesophilic enzyme pairs simply operate through different evolutionary-optimized catalytic mechanisms. In this communication, we present evidence that while the steps of the catalytic mechanisms for mesophilic and thermophilic indole-3-glycerol phosphate synthase (IGPS) enzymes are fundamentally similar, the identity of the rate-determining step changes as a function of temperature. Our findings indicate that while product release is rate-determining at 25°C for thermophilic IGPS, near its adaptive temperature (75°C), a proton transfer event, involving a general acid, becomes rate-determining. The rate-determining steps for thermophilic and mesophilic IGPS enzymes are also different at their respective, adaptive temperatures with the mesophilic IGPS-catalyzed reaction being rate-limited before irreversible CO2 release, and the thermophilic IGPS-catalyzed reaction being rate limited afterwards.     

Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria, XI. Engineering thermostability and activity of lactate dehydrogenases from bacilli

An extensive comparative structural analysis of lactate dehydrogenase (LDH) sequences from thermophilic, mesophilic and psychrophilic bacilli revealed characteristic primary structural differences. These specific amino-acid substitutions were found in the entire LDH molecule. However, in certain regions of the LDH an accumulation of these exchanges could be detected. These regions seem to be particularly important for the temperature adaptation of the enzyme. The influence of one of such regions at the N-terminus on stability and activity of LDHs was analysed by the construction of hybrid mutants between LDH sequences from thermophilic, mesophilic and psychrophilic bacilli and also by site-directed mutagenesis experiments at five different positions. The substitutions of Thr-29 or Ser-39 to Ala residues in the LDH from the mesophilic B. megaterium increased the thermostability of the enzyme drastically (15 degrees C). An increase of 20 degrees C could be observed when both amino-acid substitutions were introduced. These amino-acid substitutions resulted in an increase of Km for pyruvate and led to a three-fold reduction of the activity (kcat/Km) at 40 degrees C compared with the wild type enzyme. The influence of these amino-acid substitutions was also investigated in the LDHs from thermophilic and psychrophilic bacilli. The high heat resistance of the LDH from the thermophilic B. stearothermophilus was not altered by the Ala to Thr and Ser substitutions at positions 29 and 39, respectively. This indicates a cooperatively stabilized conformation of this LDH. However, in this mutant of the B. stearothermophilus LDH the activity (kcat/Km) was increased two-fold.     

Isolation of thermophilic mutants of Bacillus subtilis and Bacillus pumilus and transformation of the thermophilic trait to mesophilic strains

Thermophilic mutants were isolated from mesophilic Bacillus subtilis and Bacillus pumilus by plating large numbers of cells and incubating them for several days at a temperature about 10 degrees C above the upper growth temperature limit for the parent mesophiles. Under these conditions we found thermophilic mutant strains that were able to grow at temperatures between 50 degrees C and 70 degrees C at a frequency of less than 10(-10). The persistence of auxotrophic and antibiotic resistance markers in the thermophilic mutants confirmed their mesophilic origin. Transformation of genetic markers between thermophilic mutants and mesophilic parents was demonstrated at frequencies of 10(-3) to 10(-2) for single markers and about 10(-7) for two unlinked markers. With the same procedure we were able to transfer the thermophilic trait from the mutant strains of Bacillus to the mesophilic parental strains at a frequency of about 10(-7), suggesting that the thermophilic trait is a phenotypic consequence of mutations in two unlinked genes.     

Neutron scattering reveals the dynamic basis of protein adaptation to extreme temperature

To explore protein adaptation to extremely high temperatures, two parameters related to macromolecular dynamics, the mean square atomic fluctuation and structural resilience, expressed as a mean force constant, were measured by neutron scattering for hyperthermophilic malate dehydrogenase from Methanococcus jannaschii and a mesophilic homologue, lactate dehydrogenase from Oryctolagus cunniculus (rabbit) muscle. The root mean square fluctuations, defining flexibility, were found to be similar for both enzymes (1.5 A) at their optimal activity temperature. Resilience values, defining structural rigidity, are higher by an order of magnitude for the high temperature-adapted protein (0.15 Newtons/meter for O. cunniculus lactate dehydrogenase and 1.5 Newtons/meter for M. jannaschii malate dehydrogenase). Thermoadaptation appears to have been achieved by evolution through selection of appropriate structural rigidity in order to preserve specific protein structure while allowing the conformational flexibility required for activity.     

Purification and properties of pyruvate kinase from Bacillus stearothermophilus

Pyruvate kinase was purified to homogeneity from a moderate thermophile, Bacillus stearothermophilus. The molecular weight of the enzyme was found to be 250,000 on gel filtration and 242,000 on sedimentation analysis. The enzyme consisted of four identical subunits of a molecular weight of 62,000-64,000. There were no remarkable differences between the thermophilic enzyme and mesophilic enzymes in amino acid composition, secondary structure, mono- and di-valent cation requirements for activity or specificity for nucleoside diphosphates. But the thermophilic enzyme was stable at high temperature and for a longer period of storage at lower temperature. Its specific activity was relatively high even at a low temperature (30 degrees C). The enzyme exhibited homotropic positive cooperativity for phosphoenol-pyruvate, but not for ADP. It was allosterically activated by AMP, ribose 5-phosphate and nucleoside monophosphates, but not by fructose 1,6-bisphosphate. Activation by AMP and ribose 5-phosphate, and inhibition by inorganic phosphate were also observed even at the physiological temperature (60 degrees C) for the thermophile.     

Effect of process temperature on bacterial and archaeal communities in two methanogenic bioreactors treating organic household waste

The bacterial and archaeal community structure was examined in two methanogenic anaerobic digestion processes degrading organic household waste at mesophilic (37 degrees C) and thermophilic (55 degrees C) temperatures. Analysis of bacterial clone libraries revealed a predominance of Bacteroidetes (34% of total clones) and Chloroflexi (27%) at the mesophilic temperature. In contrast, in the thermophilic clone library, the major group of clones were affiliated with Thermotogae (61%). Within the domain Archaea, the phyla Euryarchaeota and Crenarchaeota were both represented, the latter only at the mesophilic temperature. The dominating archaeons grouped with Methanospirillum and Methanosarcina species at the mesophilic and thermophilic temperature, respectively. Generally, there was a higher frequency of different sequences at the lower temperature, suggesting a higher diversity compared to the community present at the thermophilic temperature. Furthermore, it was not only the species richness that was affected by temperature, but also the phylogenetic distribution of the microbial populations.     

Mirror image mutations reveal the significance of an intersubunit ion cluster in the stability of 3-isopropylmalate dehydrogenase

The comparison of the three-dimensional structures of thermophilic (Thermus thermophilus) and mesophilic (Escherichia coli) 3-isopropylmalate dehydrogenases (IPMDH, EC 1.1.1.85) suggested that the existence of extra ion pairs in the thermophilic enzyme found in the intersubunit region may be an important factor for thermostability. As a test of our assumption, glutamine 200 in the E. coli enzyme was turned into glutamate (Q200E mutant) to mimic the thermophilic enzyme at this site by creating an intersubunit ion pair which can join existing ion clusters. At the same site in the thermophilic enzyme we changed glutamate 190 into glutamine (E190Q), hereby removing the corresponding ion pair. These single amino acid replacements resulted in increased thermostability of the mesophilic and decreased thermostability of the thermophilic enzyme, as measured by spectropolarimetry and differential scanning microcalorimetry.     

Effect of molecular confinement on internal enzyme dynamics: frequency domain fluorometry and molecular dynamics simulation studies

The tryptophanyl emission decay of the mesophilic beta-galactosidase from Aspergillus oryzae free in buffer and entrapped in agarose gel is investigated as a function of temperature and compared to that of the hyperthermophilic enzyme from Sulfolobus solfataricus. Both enzymes are tetrameric proteins with a large number of tryptophanyl residues, so the fluorescence emission can provide information on the conformational dynamics of the overall protein structure rather than that of the local environment. The tryptophanyl emission decays are best fitted by bimodal Lorentzian distributions. The long-lived component is ascribed to close, deeply buried tryptophanyl residues with reduced mobility; the short-lived one arises from tryptophanyl residues located in more flexible external regions of each subunit, some of which are involved in forming the catalytic site. The center of both lifetime distribution components at each temperature increases when going from the free in solution mesophilic enzyme to the gel-entrapped and hyperthermophilic enzyme, thus indicating that confinement of the mesophilic enzyme in the agarose gel limits the freedom of the polypeptide chain. A more complex dependence is observed for the distribution widths. Computer modeling techniques are used to recognize that the catalytic sites are similar for the mesophilic and hyperthermophilic beta-galactosidases. The effect due to gel entrapment is considered in dynamic simulations by imposing harmonic restraints to solvent-exposed atoms of the protein with the exclusion of those around the active site. The temperature dependence of the tryptophanyl fluorescence emission decay and the dynamic simulation confirm that more rigid structures, as in the case of the immobilized and/or hyperthermophilic enzyme, require higher temperatures to achieve the requisite conformational dynamics for an effective catalytic action and strongly suggest a link between conformational rigidity and enhanced thermal stability.     

Structural prediction and comparative docking studies of psychrophilic ��- Galactosidase with lactose, ONPG and PNPG against its counter parts of mesophilic and thermophilic enzymes

Enzymes from psychrophiles catalyze the reactions at low temperatures with higher specific activity. Among all the psychrophilic enzymes produced, cold active β-galactosidase from marine psychrophiles revalorizes a new arena in numerous areas at industrial level. The hydrolysis of lactose in to glucose and galactose by cold active β-galactosidase offers a new promising approach in removal of lactose from milk to overcome the problem of lactose intolerance. Herein we propose, a 3D structure of cold active β-galactosidase enzyme sourced from Pseudoalteromonas haloplanktis by using Modeler 9v8 and best model was developed having 88% of favourable region in ramachandran plot. Modelling was followed by docking studies with the help of Auto dock 4.0 against the three substrates lactose, ONPG and PNPG. In addition, comparative docking studies were also performed for the 3D model of psychrophilic β-galactosidase with mesophilic and thermophilic enzymes. Docking studies revealed that binding affinity of enzyme towards the three different substrates is more for psychrophilic enzyme when compared with mesophilic and thermophilic enzymes. It indicates that the enzyme has high specific activity at low temperature when compared with mesophilic and thermophilic enzymes.