Engineering a Prokaryotic Non-P450 Hydroxylase for 3'-Hydroxylation of Flavonoids

Plant-derived cytochrome P450-dependent flavonoid 3'-hydroxylases are the rate-limiting enzymes in flavonoid biosynthesis. In this study, the large component (HpaB) of a prokaryotic 4-hydroxyphenylacetate (4-HPA) 3-hydroxylase was engineered for efficient 3'-hydroxylation of naringenin. First, we selected four HpaBs through database search and phylogenetic analysis and compared their catalytic activities toward 4-HPA and naringenin. HpaB from Rhodococcus opacus B-4 (RoHpaB) showed better preference toward naringenin. To elucidate the underlying mechanism, we analyzed the structural differences of HpaBs through homologous modeling, molecular docking, and molecular dynamics simulation, and the substrate preference of RoHpaB was mainly attributed to the shorter chain length of loop 212-222 and the larger substrate binding pocket. RoHpaB was further engineered by alanine scanning and structural replacement, and the RoHpaB^Y215A variant was obtained, whose catalytic efficiency (k_cat/K_m) toward naringenin is 25.3 times higher than that of RoHpaB. In addition, RoHpaB^Y215A also showed significantly improved activity toward flavonoids apigenin and kaempferol. This work opens the possibility of using engineered HpaB as a versatile hydroxylase to produce functionalized flavonoids.