Molecular Characterization of a Stereoselective and Promiscuous Flavanone 3-Hydroxylase from Carthamus tinctorius L

Flavanone 3-hydroxylases (F3Hs) belong to the 2-oxoglutarate-dependent dioxygenase family and play an important role in plant flavonoid biosynthesis. However, the stereoselective catalytic mechanism and substrate promiscuity of this type of enzyme are not well understood. In this study, we identified and biochemically characterized CtF3H1, an F3H from Carthamus tinctorius, a plant used in traditional Chinese medicine that exhibits high stereoselectivity and substrate promiscuity toward structurally diverse (2S)-flavanones. Isothermal titration calorimetry revealed that CtF3H1 exhibits distinctly different binding behaviors with (2S)-flavanone (2S-naringenin) and (2R)-flavanone (2R-naringenin), and these differences govern its stereoselectivity. An investigation of the structure-activity relationships between the enzyme and its substrates demonstrated that 7-OH and/or 4 '-OH are necessary for regio-and stereoselective 3-hydroxylation of (2S)-flavanones. Homology modeling and molecular docking combined with site-directed mutagenesis identified the amino acid residues necessary for hydroxylation. These findings demonstrate the potential versatility of CtF3H1 in regio-and stereohydroxylation and provide molecular insights into the catalytic mechanism of F3H for further enzyme engineering.