Directed Evolution of a high redox potential laccase

Laccases are blue multi-copper oxidoreductases catalyzing the four electron reduction of O2 to water coupled with the oxidation of small organic, mainly aromatic substrates. They have been classified as low, medium and high redox potential laccases based on equilibrium potentiometric titrations of the type 1 copper site. Only laccases from fungi, in particular from basidiomycetes, have been classified as high redox potential laccases. These enzymes are especially interesting for a large variety of biocatalytic applications as well as in the emerging field of biosensors and biofuel cells as cathode catalyst for the generation of electricity. The practical usability of these highly promising enzymes is hampered by their inhibition by hydroxide, rendering them practically inactive at pHs higher than 6.5. Despite the importance of the inhibition of high redox potential laccases by hydroxide ions and other anions (chloride, fluoride) neither the binding sites of the inhibitors nor the exact mechanisms of the inhibition have been elucidated yet. The pH profiles of the basidiomycete Trametes versicolor laccase are similar to many published laccases and are consistent with the theory of hydroxide inhibition at the T2/T3 center at near neutral pH. However, the pH profiles with different substrates of the laccases from the ascomycete Melanocarpus albomyces and the lacquer tree Rhus vernicifera disagree with a binding of OH- exclusively to the T2/T3 center. Previous research using rational protein engineering, although showing some effects on the pH behavior of laccases, did not answer the questions, where OH- ions bind, which amino acids are involved and whether a change in the pH behavior also influences inhibition by other anions. The main objective of the proposed research project is to identify amino acid positions, side chains and consequently structural properties responsible for the inhibition of fungal high redox potential laccases by hydroxide ions (pH dependency). We propose a random approach, where directed evolution will be used to find hotspots that influence the hydroxide inhibition. Semi-rational engineering will follow the directed evolution experiments to further explore the influence of these hotspots. The mutations in the laccase gene leading to an enzyme variant with a higher activity at neutral pH will be identified and a posteriori the structure-function relationship responsible for the pH behavior and the inhibition by anions in general will be elucidated. A better understanding of the structure function relationship responsible for the different pH behavior of laccases will help to engineer designer laccases with an optimal pH range for a respective application.

Laccases are enzymes that transfer electrons to oxygen with the aid of two copper centres and can thus reduce it to water. They each have a T1 centre, which consists of one copper atom and a T2/T3 centre, consisting of 3 copper atoms. Laccases with a high redox potential at this T1 centre as found in fungi can be applied as part of biosensors or biofuel cells, which could power very small devices in the future, for example in the body without needing ordinary batteries. However, laccases are not particularly suitable for conditions such as those prevailing in blood. On the one hand, they operate very slowly at neutral pH and on the other hand most of them are inhibited by chloride ions. In this project, the dependence of the activity of a laccase on the pH value as well as on the chloride ion concentration was investigated. For this purpose, the amino acid composition of the enzyme was altered by random as well as directed mutations and it was searched for enzyme variants whose pH dependence was different from the original enzyme. At the same time the variants were tested for higher stability. After the study of several tens of thousands of variants thirteen were found with altered pH behaviour and one with a higher stability. They were characterized in detail. Particular attention was paid to the relationship between amino acid structure and enzyme activity, for which the exact structure of the enzyme had been elucidated. It could be shown that the pH behaviour, unlike previously assumed, is strongly dependent on changes at the T1 copper centre, but changes in the redox potential play only a subordinate role. It was also found that chloride ions attack the T1 centre. These important findings contribute to a better understanding of the function of laccases, thus bringing them closer to future use in biobatteries.