Xingyi Zhang· Yinghui Feng · Yuanzhe Hua · Chuanxi Zhang · Bohuan Fang · Xiang Long · Yue Pan · Bei Gao · John Z. H. Zhang · Lijun Li · Hui Ni · Lujia Zhang*
The flavonoid naringenin is abundantly present in pomelo peels, and the unprocessed naringenin in wastes is not friendly for the environment once discarded directly. Fortunately, the hydroxylated product of eriodictyol from naringenin exhibits remarkable antioxidant and anticancer properties. The P450s was suggested promising for the bioconversion of the flavo noids, but less naturally existed P450s show hydroxylation activity to C3′ of the naringenin. By well analyzing the cata lytic mechanism and the conformations of the naringenin in P450, we proposed that the intermediate Cmpd I ((porphyrin) Fe = O) is more reasonable as key conformation for the hydrolyzation, and the distance between C3′/C5′ of naringenin to the O atom of CmpdI determines the hydroxylating activity for the naringenin. Thus, the “flying kite model” that gradually drags the C-H bond of the substrate to the O atom of CmpdI was put forward for rational design. With ab initio design, we successfully endowed the self-sufficient P450-BM3 hydroxylic activity to naringenin and obtained mutant M5-5, with kcat, Km, and kcat/Km values of 230.45 min−1, 310.48 µM, and 0.742 min−1 µM−1, respectively. Furthermore, the mutant M4186 was screened with kcat/Km of 4.28-fold highly improved than the reported M13. The M4186 also exhibited 62.57% yield of eriodictyol, more suitable for the industrial application. This study provided a theoretical guide for the rational design of P450s to the nonnative compounds.
