Exploring deazaflavoenzymes as biocatalysts

Enzymes are known to play important roles in our lives. Understanding how they work helps, among other things, in fighting against diseases and in the development of efficient medication. Tuberculosis affects a large population of the world. Clearly, there is a need for effective medicines against it. Recently, it was discovered that Mycobacterium tuberculosis contains the unusual cofactor F420 in higher amounts. It is known that this cofactor helps in keeping Mycobacteria alive under stress conditions. Since, this cofactor is not found in humans, F420-dependent enzymes could be good targets for combating tuberculosis. Besides potential drug targets, could F420-dependent enzymes be explored as biocatalysts, since F420 has a stronger reducing power than other flavin cofactors.
This thesis focusses on the identification and study of F420-dependent enzymes. A F420-bound column was designed to fish out novel F420-binding proteins from cell free extract. With this approach we have identified a number of potential candidate proteins using cell free extract of Mycobacterium smegmatis. We discovered a new group of F420-dependent dehydrogenases which accept a broader range of sugar-6-phosphates as substrates. Furthermore, we have identified and characterized a F420-dependent NADP+ oxidoreductase (FNO) from Thermobifida fusca. This FNO showed higher expression levels than the previously studied FNOs. An engineered version of this enzyme accepts both NAD+ and NADP+ as substrates. Our results on F420-dependent ene-reductases show that these enzymes perform reductions with high enantio- and regioselectivity. Lastly, we performed evolutionary studies on F420-dependent enzymes. These studies revealed that the FMN-dependent enzymes were the ancestors of the F420-dependent enzymes.