Molecular Dynamics of Mesophilic-Like Mutants of a Cold-Adapted Enzyme: Insights into Distal Effects Induced by the Mutations (original) (raw)
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Engineering the properties of a cold active enzyme through rational redesign of the active site
European Journal of Biochemistry, 2001
In an effort to explore the effects of local flexibility on the cold adaptation of enzymes, we designed point mutations aiming to modify side-chain flexibility at the active site of the psychrophilic alkaline phosphatase from the Antarctic strain TAB5. The mutagenesis targets were residues Trp260 and Ala219 of the catalytic site and His135 of the Mg 21 binding site. The replacement of Trp260 by Lys in mutant W260K, resulted in an enzyme less active than the wild-type in the temperature range 5-25 8C. The additional replacement of Ala219 by Asn in the double mutant W260K/A219N, resulted in a drastic increase in the energy of activation, which was reflected in a considerably decreased activity at temperatures of 5-15 8C and a significantly increased activity at 20 -25 8C. Further substitution of His135 by Asp in the triple mutant W260K/A219N/H135D restored a low energy of activation. In addition, the His135 !Asp replacement in mutants H135D and W260K/A219N/ H135D resulted in considerable stabilization. These results suggest that the psychrophilic character of mutants can be established or masked by very slight variations of the wildtype sequence, which may affect active site flexibility through changes in various conformational constraints.
Molecular BioSystems, 2012
Multiple comparison of the Molecular Dynamics (MD) trajectories of mutants in a cold-adapted a-amylase (AHA) could be used to elucidate functional features required to restore mesophilic-like activity. Unfortunately it is challenging to identify the different dynamic behaviors and correctly relate them to functional activity by routine analysis. We here employed a previously developed and robust two-stage approach that combines Self-Organising Maps (SOMs) and hierarchical clustering to compare conformational ensembles of proteins. Moreover, we designed a novel strategy to identify the specific mutations that more efficiently convert the dynamic signature of the psychrophilic enzyme (AHA) to that of the mesophilic counterpart (PPA). The SOM trained on AHA and its variants was used to classify a PPA MD ensemble and successfully highlighted the relationships between the flexibilities of the target enzyme and of the different mutants. Moreover the local features of the mutants that mostly influence their global flexibility in a mesophilic-like direction were detected. It turns out that mutations of the cold-adapted enzyme to hydrophobic and aromatic residues are the most effective in restoring the PPA dynamic features and could guide the design of more mesophilic-like mutants. In conclusion, our strategy can efficiently extract specific dynamic signatures related to function from multiple comparisons of MD conformational ensembles. Therefore, it can be a promising tool for protein engineering.
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.
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.
Journal of Bacteriology, 2005
The cold-active ␣-amylase from the Antarctic bacterium Pseudoalteromonas haloplanktis (AHA) is the largest known multidomain enzyme that displays reversible thermal unfolding (around 30°C) according to a two-state mechanism. Transverse urea gradient gel electrophoresis (TUG-GE) from 0 to 6.64 M was performed under various conditions of temperature (3°C to 70°C) and pH (7.5 to 10.4) in the absence or presence of Ca 2؉ and/or Tris (competitive inhibitor) to identify possible low-stability domains. Contrary to previous observations by strict thermal unfolding, two transitions were found at low temperature (12°C). Within the duration of the TUG-GE, the structures undergoing the first transition showed slow interconversions between different conformations. By comparing the properties of the native enzyme and the N12R mutant, the active site was shown to be part of the least stable structure in the enzyme. The stability data supported a model of cooperative unfolding of structures forming the active site and independent unfolding of the other more stable protein domains. In light of these findings for AHA, it will be valuable to determine if active-site instability is a general feature of heat-labile enzymes from psychrophiles. Interestingly, the enzyme was also found to refold and rapidly regain activity after being heated at 70°C for 1 h in 6.5 M urea. The study has identified fundamental new properties of AHA and extended our understanding of structure/stability relationships of cold-adapted enzymes.
Biochimica Et Biophysica Acta-proteins and Proteomics, 2006
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.
Proteins-structure Function and Bioinformatics, 2006
The cold-adapted α-amylase from Pseudoalteromonas haloplanktis (AHA) is a multidomain enzyme capable of reversible unfolding. Cold-adapted proteins, including AHA, have been predicted to be structurally flexible and conformationally unstable as a consequence of a high lysine-to-arginine ratio. In order to examine the role of low arginine content in structural flexibility of AHA, the amino groups of lysine were guanidinated to form homo-arginine (hR), and the structure–function–stability properties of the modified enzyme were analyzed by transverse urea gradient-gel electrophoresis. The extent of modification was monitored by MALDI-TOF-MS, and correlated to changes in activity and stability. Modifying lysine to hR produced a conformationally more stable and less active α-amylase. The kcat of the modified enzyme decreased with a concomitant increase in ΔH# and decrease in Km. To interpret the structural basis of the kinetic and thermodynamic properties, the hR residues were modeled in the AHA X-ray structure and compared to the X-ray structure of a thermostable homolog. The experimental properties of the modified AHA were consistent with K106hR forming an intra-Domain B salt bridge to stabilize the active site and decrease the cooperativity of unfolding. Homo-Arg modification also appeared to alter Ca2+ and Cl− binding in the active site. Our results indicate that replacing lysine with hR generates mesophilic-like characteristics in AHA, and provides support for the importance of lysine residues in promoting enzyme cold adaptation. These data were consistent with computational analyses that show that AHA possesses a compositional bias that favors decreased conformational stability and increased flexibility. Proteins 2006. © 2006 Wiley-Liss, Inc.
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.
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.
Dynamic Properties of a Psychrophilic α-Amylase in Comparison with a Mesophilic Homologue
Journal of Physical Chemistry B, 2009
The cold-active, chloride-dependent R-amylase from Pseudoalteromonas haloplanktis (AHA) is one of the best characterized psychrophilic enzymes, and shares high sequence and structural similarity with its mesophilic porcine counterpart (PPA). An atomic detail comparative analysis was carried out by performing more than 60 ns of multiple-replica explicit-solvent molecular dynamics simulations on the two enzymes in order to characterize the differences in ensemble properties and dynamics in solution between the two homologues. We find in both enzymes high flexibility clusters in the surroundings of the substrate-binding groove, primarily involving the long loops that protrude from the main domain's barrel structure. These loops are longer in PPA and extend further away from the core of the barrel, where the active site is located: essential fluctuations in PPA mainly affect the highly solvent-accessible portions of these loops, whereas AHA is characterized by greater flexibility in the immediate surroundings of the active site. Furthermore, detailed analysis of activesite dynamics has revealed that elements previously identified through X-ray crystallography as involved in substrate binding in both enzymes undergo concerted motions that may be linked to catalysis.