Temperature Adaptation By Sitedirected Mutagenesis And Directed Evolution

Evolution of a particular trait necessitates that a single base change can at times lead to a single amino acid substitution that brings about alterations in a protein's properties, which is beneficial to the host. Site-directed mutagenesis is therefore often employed in tandem with rational design ideas in order to understand the functioning of enzymes. Theories derived from the study of a three-dimensional model of a particular enzyme are utilized to select particular residues for mutation and predict the possible outcome. Thus, experiments with T4 lysozyme showed that even a single mutation in the hydrophobic core of an enzyme can have dramatic effects on stability (138). This may be informative, but it does not necessarily cover all the parts of the molecule that can affect its properties. Attention is often directed at the active-site region and at internal residues, whereas more distant residues may cause functional alterations, even those that reside on the outside surface of the molecule.

Studies using site-directed mutagenesis, however, have uncovered the fact that thermostability is not systematically inversely related to specific activity, one example being subtilisin excreted by an Antarctic Bacillus TA39 (139). The enzyme displays the usual characteristics of cold-active enzymes—i.e., a high catalytic efficiency at low temperatures and an increased thermosensitivity. The affinity for calcium is also almost 3 orders of magnitude lower than that of meso-philic subtilisins. An important stabilization of the molecular structure was achieved through a modification of one residue acting as a calcium ligand. The thermostability of the mutated product expressed in a mesophilic Bacillus reached that of mesophilic subtili-sin, and this mutation further enhanced the specific activity by a factor close to 2 when compared to the wild-type enzyme.

Single-base mutations do not cover a large fraction of protein sequence space since they often produce conservative substitutions. Another approach that increases nonconservative substitutions is that of directed evolution, where rapid screening procedures are combined with random mutagensis and in vitro recombination. Structural analysis of selected mutants can then bring about understanding of the observed phe-notype, be it stability or catalytic activity, in terms of chemical function. The vast majority of possible evolutionary paths lead to poorer enzymes, so for successful directed evolution, the strategic challenge is to choose the right path that will eventually improve the desired features (140).

Recent examples can be found where the stability of enzymes from psychrophiles or mesophiles has been increased without effects on activity. A moderately stable thermolysin-like metalloprotease from Bacillus stearothermophilus was made hyperstable by a limited number of mutations. An eightfold mutant enzyme had a half-life of 2.8h at 100°C, but still displayed wild-type-like activity at 37°C (24, 141). Subtilisin E from a mesophilic Bacillus subtilis was converted into an enzyme functionally equivalent to its thermophilic counterparts by directed evolution (142). Subtilisin E differs from thermitase at 157 amino acid positions. However, only eight amino acid substitutions were sufficient to convert subtilisin E into an enzyme equally thermostable, a result from five generations of DNA alterations. Interestingly, the eight substitutions were distributed over the surface of the enzyme. Only two of those are found in thermitase. Also, thermostability could be increased without compromising enzyme activity (142).

In another study, thermoresistance was engineered into bacterial p-glucosidase by a directed-evolution strategy (143), whereas no significant alterations in kinetic parameters were observed. The main factors for increasing the thermostability were a combination of one extra salt bridge, replacement of a solventexposed asparagine residue, stabilization of the hydro-phobic core, and stabilization of the quaternary structure. An earlier study used in vitro evolution to probe the relationship between stability and activity in a mesophilic esterase. Six generations of random muta-genesis, recombination, and screening, stabilized Bacillus subtilis p-nitrobenzyl esterase significantly without compromising its catalytic activity at lower temperatures. It was also found that mutations that increased thermostability, while maintaining low-tem perature activity, were very rare. The results suggested that stability at high temperatures was not incompatible with high catalytic activity at low temperatures because of perceived mutually exclusive demands on enzyme flexibility (144).

Low-temperature activity can also be generated by mutations. It was possible to improve catalytic activity at 37°C of a thermophilic enzyme with a single or double amino acid substitution. DNA shuffling was used to mutate indoleglycerol phosphate synthase from the hyperthermophile Sulfolobus solfataricus (145). The parental enzyme's turnover number at room temperature was limited by the dissociation of the enzyme-product complex, apparently because the loops that obstruct the active site were not flexible enough at low temperatures. In the variants, the binding and release of product was much more rapid, and this shifted the rate-determining step to a preceding chemical step (145). Similarly, multiple random mutants of p-glucosidase from the hyperthermophile Pyrococcus furiosus were screened for increased activity at room temperature (146). Multiple variants were identified with up to threefold increased rates of substrate hydrolysis. Amino acid substitutions were widespread, occurring in the active-site region, at the enzyme surface, buried in the interior of the monomers, and at subunits interfaces. Interestingly, low-temperature activity was achieved in different ways, by altering substrate specificity or by overall destabli-zation of the enzyme. Single amino acid substitutions were sufficient to drastically alter the kinetic properties, as would be expected, if evolutionary processes are to work. The enzyme was able to accommodate in its interior amino acids with larger or smaller side chains, and with different properties without affecting thermo-stability. Substrate specificity was also determined by substitutions distant to the active site.

The stability of enzymes from psychrophiles can also be increased without reducing activity (25). The psychrophilic protease subtilisin S41 was subjected to two different selection pressures. The evolved subtili-sin S41 retained its psychrophilic character as a catalyst in spite of its dramatically increased thermostability. These results demonstrated that it is possible to increase activity at low temperatures and stability at high temperatures simultaneously. It was concluded that the fact that enzymes displaying both properties are not found in nature, most likely reflects the effects of evolution rather than any intrinsic physico-chemical limitations on proteins (25). However, the dependence of slower thermal inactivation on calcium concentration indicated that enhanced calcium binding was largely responsible for the increased stability.

Few studies are to be found where the kinetic characteristics of a psychrophilic enzyme have been produced by directed evolutionary methodology. A cold-adapted subtilisin has been generated by evolutionary based sequential methodology. Cold-adaptation was achieved with three mutations, and it was found that an increase in substrate affinity (i.e., decreased Km value) was mostly responsible for the observed doubling in catalytic efficiency at 10°C (147).

This section has shown that single-residue substitutions can affect stability of enzymes considerably and influence kinetic properties. However, it may be generally a fact that more than one substitution is required to realize the full potential for temperature adaptation in an enzyme.

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