Redirecting a Fungal Quercetin 2,3‐Dioxygenase Toward Artificial Flavonols

ABSTRACT A novel quercetin 2,3‐dioxygenase from Penicillium chrysogenum , following biochemical characterization, served as the starting point for reshaping the substrate‐binding cavity to alter its substrate specificity. Using a rational engineering strategy supported by computational predictive tools, we achieved high activity toward specific artificial flavonols. In all generated variants, amino acids were replaced with residues that naturally occur at the selected positions in homologous enzymes. Two variants with enlarged substrate‐binding cavities exhibited improved activity toward bulkier substrates. In particular, the Y55F‐F134L‐M143L variant showed 20‐ to 1750‐fold higher activity toward flavonol compounds with phenyl‐based substitutions at position C‐8. Conversely, one variant with a smaller substrate‐binding cavity showed 15‐fold higher activity toward the smaller flavonol 3,7‐dihydroxyflavone. The procedure described here has implications for engineering metalloenzymes to alter their substrate specificity toward novel compounds.