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FRINATEK-Fri prosj.st. mat.,naturv.,tek

Evolutionary Principles of Biocatalysts From Extreme Environments

Alternative title: Biokatalysatorer fra ekstreme miljø - evolusjonære prinsipp

Awarded: NOK 12.9 mill.

How organisms can survive and thrive in environments not typically considered compatible with life, such as temperatures above the boiling point and below the freezing point of water, has been challenging to fully understand. Through millions of years of adaptation, organisms have used different strategies to survive in such harsh environments. Atomic and moleculare motions are significantly reduced around the freezing point of water, and the rate of chemical reactions decreases exponentially when the temperature is lowered. Cold-adapted organisms can counteract this by synthesizing specialized enzyme that catalyze the chemical reaction very efficiently at low to moderate temperatures. What enables these enzymes to function at low temperatures where their warm-active counterparts are inactive? Comparison of the three dimensional structure of homologous enzymes looks at first sight very similar, and the active site where the chemical reaction takes place is in most cases identical. The difference in biological activity seems to arise from structural differences distant to the active site. The main hypothesis currently pursued is that the difference in biological activity between cold- and warm-active enzymes is due to a softer protein surface for enzymes operating at low temperatures. A softer surface is then believed to reduce the amount of energy required to bring the reactants to products in the reactions catalyzed by cold-active enzymes compared to their warm-active homologues. Several enzymes from closely related organisms have been chosen in order examine our main hypothesis. This includes so far lipase, esterase, chorismate mutates, amylase and endonuclease. We have established protocols and systems to produce and characterize these enzymes in the laboratory. Through comparative studies where cold-active enzymes are compared with the warm-active counterparts, catalytic activity as a function of temperature and the sensitivity to thermal melting have been carried out. Furthermore, the three dimensional structure of several of these enzymes has been solved using x-ray crystallography, which is pivotal for further studies with molecular modeling techniques. Cold-adapted enzymes are characterized both by a higher catalytic activity at low temperatures and by having their temperature optimum down-shifted, compared to mesophilic orthologs. In several cases, the optimum does not coincide with the onset of protein melting but reflects some other type of inactivation. In the psychrophilic a-amylase from an Antarctic bacterium, the inactivation is thought to originate from a specific enzyme-substrate interaction that breaks around room temperature. We have used computational techniques to redesign this enzyme with the aim at shifting its temperature optimum upward. A set of mutations designed to stabilize the enzyme-substrate interaction were predicted by computer simulations of the catalytic reaction at different temperatures. The predictions were verified by kinetic experiments and crystal structures of the redesigned a-amylase, showing that the temperature optimum is indeed markedly shifted upward and that the critical surface loop controlling the temperature dependence approaches the target conformation observed in a mesophilic ortholog. An interesting question to ask in this relation is whether nature has evolved something similar to our designed enzymes. Searching through protein sequence databases revealed that our designed enzyme is most closely related to a corresponding enzyme from a warm-adapted bacteria. It thus appears that natural evolution may have been working along the same lines as our computational strategy. The project also focused on the development of software that lowers the threshold for applying advanced computational techniques by using graphical user interfaces to set up, run and analyze quantum mechanical calculations.

Our planet has several environments that are potentially hostile to life. The survival of organisms has required the expression of proteins adapted to function under extreme temperature, pH, pressure and ionic strength. However, the origin of such adaptations is in most case still an open question. Faced with an exponential decrease in chemical reaction rates as the temperature is lowered, cold-adapted organisms require specialized enzymes to maintain a functional metabolism. Although often highly homologous to their mesophilic counterparts, these enzymes have some characteristic and universal properties that reflect their evolutionary optimization. Such enzymes catalyze their reactions with lower activation enthalpies counterbalanced by more negative entropies, yielding higher rates at low temperatures. The structural origin of the seemingly universal change in the activation enthalpy-entropy balance for cold-adapted enzymes remains very puzzling. The basic problem with connecting macroscopic thermodynamic quantities, such as and derived from experimental Arrhenius plots, to the 3D protein structure is that the underlying microscopic energetics is essentially inaccessible to experiment. Extensive computer simulations and calculated high-precision Arrhenius plots may now offer a solution to reveal the evolutionary principles involved in tuning the enthalpy-entropy balance. The interest in enzymes from extremophiles (extremozymes) from the viewpoint of industrial and biotechnological applications is also immense, as their potential use as biocatalysts may offer many advantages due to the fact that their activity is tailored for unusual environmental conditions. Such biocatalysts may either be exploited directly or be reengineered from orthologous mesophilic enzymes, given that the design principles of the desired biophysical properties are known.

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FRINATEK-Fri prosj.st. mat.,naturv.,tek