PhD defence S. (Sara) Russo

Exploration and engineering of a nitroreductase for sustainable pharmaceutical synthesis

Nitroreductases (NRs) are NAD(P)H-dependent flavoenzymes that convert nitroaromatic compounds into arylamines via the nitroso and hydroxylamine intermediates. They use a non-covalently bound FMN as prosthetic group and a nicotinamide cofactor, NAD(P)H, as the electron source. Because of their broad substrate scope, these enzymes have found applications in multiple fields such as bioremediation, cell imaging, and prodrug activation. Nonetheless, in recent years, the versatility of nitroreductases has expanded to include the synthesis of highly valuable pharmaceutical compounds such as aromatic and aliphatic amines, azoxy and azobenzenes, as well as N-heterocycles. Our goal was to expand the existing toolbox of nitroreductases for the efficient synthesis of pharmaceutically relevant arylamines and to gain a better understanding of their catalytic mechanism and substrate scope. This thesis discusses the most recent advances in nitroreductases as biocatalysts for amine formation (Chapter 1) and provides a comprehensive characterization of a novel nitroreductase from Bacillus tequilensis, named BtNR. The substrate scope of this enzyme (Chapter 2), as well as its biochemical, kinetic, and structural properties (Chapter 3), were thoroughly described. Furthermore, BtNR was converted into a covalent flavoprotein using the Flavin-fixing method (Chapter 4). The effects of different flavinylation attachment sites on the biochemical and kinetic properties of the flavinylated BtNR variants were investigated (Chapter 5).