Flavoproteins are prevalent in nature, and they are involved in many biochemical reactions. Flavoproteins contain a non-, mono- or bicovalently attached FMN or FAD cofactor for catalysis. The berberine bridge enzyme (BBE) is a central enzyme in benzylisoquinoline alkaloid biosynthesis and catalyzes the conversion of (S)-reticuline to (S)-scoulerine. BBE possesses a bicovalently linked FAD with a covalent linkage between the 8?-position of the flavin ring system and His104 and between the 6-position and Cys166. Trp165 was identified as an important residue in the substrate binding site of BBE, which shows a high degree of flexibility and which might be crucial for substrate specificity of the enzyme. Here, I present a detailed biochemical and structural characterization of the BBE variant W165F from Eschscholzia californica (California poppy), where I especially tried to investigate the influence of this amino acid on catalysis as well as on the structural organization of the active site. Trp165 was replaced with phenylalanine using site-directed mutagenesis and the variant protein was structurally and biochemically characterized. The determined kinetic parameters like turnover rate, redox potential, reductive and oxidative rate are comparable to the wild type enzyme. Additionally, a degradation of the flavin to a 4a-spirohydantoin was observed. Again this behavior was already known for the wild type enzyme. From the crystal structure of the BBE W165F variant it is obvious that the amino acid exchange with phenylalanine might provide more space for the conversion of even larger substrates. However, studies with substrate analogs still have to be performed to verify this hypothesis. Moreover, the activity of BBE towards new substrates can be improved by protein engineering techniques, thus new BBE mutants were created, where Trp165 was replaced with small amino acids to hopefully provide even more space for bulky substrates (W165A, W165V and W165L).