Abstract
Metalloenzymes are a class of enzymes that catalyze through the metal ions they load. Angelica archangelica flavone synthase I (AnFNS I) and Camellia sinensis flavonol synthase (CaFLS), both of which belong to metalloenzymes, have highly similar structures and metal catalytic cores. However, these two enzymes catalyze the same substrate to produce significantly different products. To identify the cause for the differences in the catalytic characteristics of AnFNS I and CaFLS, their protein models were constructed using homology modeling. Structural alignment and molecular docking was also used to elucidate the molecular basis of the differences observed. To analyze and verify the cause for the differences in the catalytic characteristics of AnFNS I and CaFLS, partial fragments of AnFNS I were used to replace the corresponding fragments on CaFLS, and the catalytic characteristics of the mutants were determined by bioconversion assay in E. coli and in vitro catalytic test. The results suggest that the difference in catalytic characteristics between AnFNS I and CaFLS is caused by the depth of the active pockets and the relative position of the substrate. Mutant 10 which present similar dock result with AnFNS I increased the proportion of diosmetin (a flavone) from 2.54 to 16.68% and decreased the proportion of 4'-O-methyl taxifolin (a flavanol) from 47.28 to 2.88%. It was also indicated that the atoms in the substrate molecule that determine the catalytic outcome may be H-2 and H-3, rather than C-2 and C-3. Moreover, it is speculated that the change in the catalytic characteristics at the changes relative spatial position of H-2/H-3 of hesperetin and the loaded carbonyl iron, caused by charged residues at the entrance of the active pocket, is the key factor for the biosynthesis of flavone from flavanone.