Engineering the properties of a cold active enzyme through rational redesign of the active site (original) (raw)
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Directed evolution study of temperature adaptation in a psychrophilic enzyme
Journal of Molecular Biology, 2000
We have used laboratory evolution methods to enhance the thermostability and activity of the psychrophilic protease subtilisin S41, with the goal of investigating the mechanisms by which this enzyme can adapt to different selection pressures. A combined strategy of random mutagenesis, saturation mutagenesis and in vitro recombination (DNA shuf¯ing) was used to generate mutant libraries, which were screened to identify enzymes that acquired greater thermostability without sacri®cing lowtemperature activity. The half-life of seven-amino acid substitution variant 3-2G7 at 60 C is 500timesthatofwild−typeandfarsurpassesthoseofhomologousmesophilicsubtilisins.Thedependenceofhalf−lifeoncalciumconcentrationindicatesthatenhancedcalciumbindingislargelyresponsiblefortheincreasedstability.Thetemperatureoptimumoftheactivityof3−2G7isshiftedupwardby500 times that of wild-type and far surpasses those of homologous mesophilic subtilisins. The dependence of half-life on calcium concentration indicates that enhanced calcium binding is largely responsible for the increased stability. The temperature optimum of the activity of 3-2G7 is shifted upward by 500timesthatofwild−typeandfarsurpassesthoseofhomologousmesophilicsubtilisins.Thedependenceofhalf−lifeoncalciumconcentrationindicatesthatenhancedcalciumbindingislargelyresponsiblefortheincreasedstability.Thetemperatureoptimumoftheactivityof3−2G7isshiftedupwardby10 C. Unlike natural thermophilic enzymes, however, the activity of 3-2G7 at low temperatures was not compromised. The catalytic ef®ciency, k cat /K M , was enhanced $threefold over a wide temperature range (10 to 60 C). The activation energy for catalysis, determined by the temperature dependence of k cat /K M in the range 15 to 35 C, is nearly identical to wild-type and close to half that of its highly similar mesophilic homolog, subtilisin SSII, indicating that the evolved S41 enzyme retained its psychrophilic character in spite of its dramatically increased thermostability. These results demonstrate that it is possible to increase activity at low temperatures and stability at high temperatures simultaneously. The fact that enzymes displaying both properties are not found in nature most likely re¯ects the effects of evolution, rather than any intrinsic physicalchemical limitations on proteins.
Current Protein & Peptide Science, 2011
The identification of molecular mechanisms underlying enzyme cold adaptation is a hot-topic both for fundamental research and industrial applications. In the present contribution, we review the last decades of structural computational investigations on cold-adapted enzymes in comparison to their warm-adapted counterparts. Comparative sequence and structural studies allow the definition of a multitude of adaptation strategies. Different enzymes carried out diverse mechanisms to adapt to low temperatures, so that a general theory for enzyme cold adaptation cannot be formulated. However, some common features can be traced in dynamic and flexibility properties of these enzymes, as well as in their intra-and inter-molecular interaction networks. Interestingly, the current data suggest that a family-centered point of view is necessary in the comparative analyses of cold-and warm-adapted enzymes. In fact, enzymes belonging to the same family or superfamily, thus sharing at least the three-dimensional fold and common features of the functional sites, have evolved similar structural and dynamic patterns to overcome the detrimental effects of low temperatures.
PLOS One, 2011
Networks and clusters of intramolecular interactions, as well as their ''communication'' across the three-dimensional architecture have a prominent role in determining protein stability and function. Special attention has been dedicated to their role in thermal adaptation. In the present contribution, seven previously experimentally characterized mutants of a cold-adapted a-amylase, featuring mesophilic-like behavior, have been investigated by multiple molecular dynamics simulations, essential dynamics and analyses of correlated motions and electrostatic interactions. Our data elucidate the molecular mechanisms underlying the ability of single and multiple mutations to globally modulate dynamic properties of the cold-adapted a-amylase, including both local and complex unpredictable distal effects. Our investigation also shows, in agreement with the experimental data, that the conversion of the cold-adapted enzyme in a warm-adapted variant cannot be completely achieved by the introduction of few mutations, also providing the rationale behind these effects. Moreover, pivotal residues, which are likely to mediate the effects induced by the mutations, have been identified from our analyses, as well as a group of suitable candidates for protein engineering. In fact, a subset of residues here identified (as an isoleucine, or networks of mesophilic-like salt bridges in the proximity of the catalytic site) should be considered, in experimental studies, to get a more efficient modification of the features of the cold-adapted enzyme.
Journal of Biological Chemistry, 2011
Background: Cold-adapted enzymes remain catalytically active at low temperatures. Results: Mutants of a cold-adapted ␣-amylase stabilized by engineered weak interactions and a disulfide bond have lost the kinetic optimization to low temperatures. Conclusion: The disappearance of stabilizing interactions in psychrophilic enzymes increases the dynamics of active site residues at low temperature, leading to a higher activity. Significance: An experimental support to the activity-stability relationships.
Journal of Molecular Biology, 2010
Molecular aspects of thermal adaptation of proteins were studied by following the co-evolution of temperature dependence, conformational stability, and substrate specificity in a cold-active lipase modified via directed evolution. We found that the evolution of kinetic stability was accompanied by a relaxation in substrate specificity. Moreover, temperature dependence and selectivity turned out to be mutually dependent. While the wild-type protein was strictly specific for short-chain triglycerides (C4) in the temperature range 10-50°C and displayed highest activity in the cold, its stabilized variant was able to accept C8 and C12 molecules and its selectivity was temperature dependent. We could not detect any improvement in the overall structural robustness of the mutant when the structure was challenged by temperature or chemical denaturants. There is, however, strong evidence for local stabilization effects in the active-site region provided by two independent approaches. Differential scanning fluorimetry revealed that the exposure of hydrophobic patches (as the active site is) precedes denaturation, and molecular dynamics simulations confirmed that stability was obtained by restriction of the mobility of the lid, a flexible structure that regulates the access to the enzyme active site and influences its stability. This reduction of lid movements is suggested to be accompanied by a concomitant increase in the mobility of other protein regions, thus accounting for the observed broadening of substrate specificity.
Psychrophilic enzymes: a thermodynamic challenge
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1997
Psychrophilic microorganisms, hosts of permanently cold habitats, produce enzymes which are adapted to work at low temperatures. When compared to their mesophilic counterparts, these enzymes display a higher catalytic efficiency over a temperature range of roughly 0-308C and a high thermosensitivity. The molecular characteristics of cold enzymes originating from Antarctic bacteria have been approached through protein modelling and X-ray crystallography. The deduced three-dimensional structures of cold a-amylase, b-lactamase, lipase and subtilisin have been compared to their mesophilic homologs. It appears that the molecular adaptation resides in a weakening of the intramolecular interactions, and in some cases in an increase of the interaction with the solvent, leading to more flexible molecular edifices capable of performing catalysis at a lower energy cost. q 1997 Elsevier Science B.V.
Journal of Biological Chemistry, 2001
3-Isopropylmalate dehydrogenase (IPMDH) from the psychrotrophic bacterium Vibrio sp. I5 has been expressed in Escherichia coli and purified. This coldadapted enzyme is highly homologous with IPMDHs from other organisms, including mesophilic E. coli and thermophilic Thermus thermophilus bacteria. Its molecular properties are similar to these counterparts. Whereas the E. coli and T. thermophilus enzymes are hardly active at room temperature, the Vibrio IPMDH has reasonable activity below room temperature. The thermal stabilities, conformational flexibilities (hydrogen-deuterium exchange), and kinetic parameters of these enzymes were compared. The temperature dependence of the catalytic parameters of the three enzymes show similar but shifted profiles. The Vibrio IPMDH is a much better enzyme at 25°C than its counterparts. With decreasing temperature i.e. with decreasing conformational flexibility, the specific activity reduces, as well; however, in the case of the Vibrio enzyme, the residual activity is still high enough for normal physiological operation of the organism. The cold-adaptation strategy in this case is achieved by creation of an extremely efficient enzyme, which has reduced but still sufficient activity at low temperature.
Taking Advantage of Promiscuity of Cold-Active Enzymes
Applied Sciences
Cold-active enzymes increase their catalytic efficiency at low-temperature, introducing structural flexibility at or near the active sites. Inevitably, this feat seems to be accompanied by lower thermal stability. These characteristics have made cold-active enzymes into attractive targets for the industrial applications, since they could reduce the energy cost in the reaction, attenuate side-reactions, and simply be inactivated. In addition, the increased structural flexibility could result in broad substrate specificity for various non-native substrates, which is called substrate promiscuity. In this perspective, we deal with a less addressed aspect of cold-active enzymes, substrate promiscuity, which has enormous potential for semi-synthesis or enzymatic modification of fine chemicals and drugs. Further structural and directed-evolutional studies on substrate promiscuity of cold-active enzymes will provide a new workhorse in white biotechnology.
Cold-active enzymes and their applications in industrial fields -A review
Cold-active enzymes are produced by organisms adapted to permanently cold habitats. Due to the depressive effect of low temperatures on reaction rates, these enzymes have to be adapted to secure appropriate reaction rates in those organisms that often thrive in environments characterized by temperatures close or below the freezing point of water. They are encountered in all prokaryotic or eukaryotic organisms adapted to cold such as microorganisms, invertebrates, insects and fish originating from the Arctic and Antarctic zones, as well as from alpine regions, glaciers or permafrost zones. They are characterized by a high specific activity at low temperatures, in any case higher than that of their mesophilic and thermophilic counterparts. This higher specific activity is generally accompanied by a decrease in thermal stability illustrated by a shift of the apparent optimum towards low temperatures, and by an important decrease in the thermodynamic stability characterized by a significantly lower stabilization enthalpy. The generally low stability induces an increase in the flexibility of the overall edifice or of crucial zones for activity of the molecular structure. There is apparently a continuum in the adaptation since some enzymes display extreme adaptation illustrated by a severe shift of the activity towards low temperatures whereas others are moderately adapted. This probably depends on their position in a metabolic pathway, on their intracellular or extracellular localization, on the environmental temperature and on the evolutionary history of the organisms.