Screening Reconstruction And Mechanism Research Of Flavonoid Hydroxylase HpaBC

Flavonoids are polyphenolic compounds that are widely present in plants.They have many biological activities such as antioxidant,antibacterial,anti-inflammatory and liver protection.They are widely used in food and medicine fields.The ortho-hydroxylation reaction is an important modification reaction,catalyzed by oxygenases,which can enhance the activity of flavonoids.This reaction is mainly catalyzed by P450 enzyme,but P450 is a membrane protein,which is poorly soluble and difficult to express in prokaryotes.Therefore,the development of monooxygenases that can be highly expressed in prokaryotes has important research value for the biosynthesis of flavonoids.In this study,a monooxygenase Ro Hpa B^Y215A,which can efficiently catalyze the o-hydroxylation of flavonoids,was obtained through screening,structural analysis and reconstruction.Firstly,Hpa B derived from E.coli,R.opacus,K.Pneumoniae and P.putida were selected through genome mining and phylogenetic tree analysis.Their catalytic activities towards p-hydroxyphenylacetic acid（4HPA）and naringenin were compared by in vitro enzyme activity assays.The results showed that Ro Hpa B had obvious substrate preference for naringenin compared with the other three enzymes,and the k_cat/K_mfor naringenin was240.80 s^-1M^-1.Adding naringenin to in vivo culture for 24 h can catalyze the production of 78.37 mg/L eriodictyol.The structural differences of Hpa B were further analyzed by structural modeling,molecular docking and kinetic simulation.It was found that the chain length of Loop212-222 located at the entrance of the substrate binding pocket of Ro Hpa B was significantly shorter than that of Ec Hpa B and Kp Hpa B.The volume of the substrate binding pocket was measured and found Ro Hpa B is indeed larger than that of Ec Hpa B and Kp Hpa B,which may be the key factor that Ro Hpa B can better accommodate naringenin.The loop region was modified by structural replacement and alanine scanning,and the amino acid residues at a distance of 8（?）near the substrate binding pocket were modified by saturation mutation,and finally an engineered enzyme with high catalytic activity Ro Hpa B^Y215Awas obtained.The catalytic efficiency of Ro Hpa B^Y215Afor naringenin was 6360.95 s^-1M^-1,which was 26.42 times higher than that of Ro Hpa B.Adding naringenin,the highest yield of eriodictyol was 414.93 mg/L.It is the highest yield of eriodictyol reported by biotransformation in E.coli.Ro Hpa B^Y215Awas applied to the hydroxylation of flavones apigenin,flavonols kaempferol and isoflavonoids genistein.The catalytic efficiency of Ro Hpa B^Y215Afor genistein(1877.93 s^-1M^-1)was 23.93 times higher than that of Ro Hpa B(78.46 s^-1M^-1).The catalytic efficiency of Ro Hpa B^Y215Awas 15.58 s^-1M^-1for apigenin and10.59 s^-1M^-1for kaempferol,while the catalytic efficiency of Ro Hpa B for apigenin and kaempferol was too low to be detected.Furthermore,this is the first time that Hpa B is used for the hydroxylation of apigenin and genistein,which expands the substrate specificity of Hpa B.The ortho-hydroxylation activity of Ro Hpa B^Y215Aobtained in this study on flavonoids was significantly improved,which provided an efficient biological element for the biosynthesis of flavonoid derivatives.