Semi-rational Engineering Of P450 BM3 And Its Application In Hydroxylation Of LCA

In the field of traditional Chinese medicine and modern medicine,bile acids play the important roles due to their various biological activity,especially in treatment of hepatobiliary diseases.The specific structures with different hydroxy substituents(the number,position and the stereochemistry)lead to the distinct difference in the solubility,function and pharmacodynamics.For example,lithocholic acid(LCA)is a secondary bile acid with very low biological activity,while ursodeoxycholic acid(UDCA)as the 7β-hydroxylation product of LCA has been approved for the treatment of primary biliary cirrhosis(PBC),but chenodeoxycholic acid(CDCA)as the 7α-isomer of UDCA features much lower biological activity than UDCA and cannot be in clinic.Therefore,the research of hydroxylation of LCA plays an important role in increasing the diversity of bile acids.In this thesis,the P450 BM3 mutant(LG-23)was selected as the initial research object and the LCA was used as substrate.Then a highly active mutant with complete regio-and stereo-selectivity for 1β-hydroxylation of LCA was obtained after four rounds of screening.The main research contents and results were as follows:1.CYP3A4,CYP7A1,Olep mutant(F84Q/S240A/V291G)and P450 BM3 mutant(LG23)were successfully expressed and their hydroxylation activity to LCA were detected.Highperfoemance liquid chromatography(HPLC)results showed that no hydroxylation products were found in CYP3A4 and CYP7A1;the Olep mutant(F84Q/S240A/V291G)exhibited only 6.0%conversion of LCA;LG-23 did 13.9%conversion of LCA.LG-23 was finally selected as the starting template due to its high expression and self-sufficient property,as well as its relatively good catalytic activity towards LCA.2.The G87 position of LG-23 was first mutated to more hydrophobic amino acids(alanine and valine).As a result,the mutants G87A and G87V increased the conversion rate of LCA by 3.0 and 2.0 folds,respectively.In addition,a new hydroxylation product was generated in the G87A-catalyzed hydroxylation of LCA and obtained by preparative enzyme-catalyzed reaction.The product was purified by silica gel and unambiguously identified to be 1β,3α-dihydroxy5β-cholanic acid(1β-OH-LCA)by NMR,high resolution mass spectrometry and single-crystal X-ray diffraction.3.The crystal structure of LG-23(61y4)was analyzed by Hotspot Wizard and Proteins Plus online servers and the docking structure of mutant G87A with LCA was further studied.Then,11 key residues were identified around the substrate channel and binding pocket for further mutation.After four rounds of design,the best mutant G87A/W72T/A74L/L181M was identified to be as high selectivity as 99.4%and 68.1%conversion of LCA.4.Finally,the effect of mutation on the activity and selectivity of LCA hydroxylation was investigated.The enzymatic kinetic parameters of template LG-23 and mutants G87A,G87A/W72T,G87A/W72T/A74L and G87A/W72T/A74L/L181M were determined.The results showed that the binding ability and the catalytic efficiency of the optimal mutant G87A/W72T/A74L/L181M to LCA were weakened compared to mutant G87A,but the production rate of 1β-OH-LCA increased by 7.5 folds.In addition,the turnover number(TON)of G87A/W72T/A74L/L181M was increased by nearly 26 folds compared to the template LG23.Molecular docking studies provided a theoretical basis for the regio-and stereo-selectivity of this reaction:after four rounds of mutations,the distance between the C1 atom of LCA and the heme iron oxygen atom was significantly shortened from 4.6 (?) to 3.2 (?),which could be one of the important reasons for the increased catalytic efficiency of LCA hydroxylati on.By analyzing the hydrogen bonding interaction between LCA and surrounding amino acids residues,it was found that in the mutants G87A/W72T,G87A/W72T/A74L and G87A/W72T/A74L/L181M,one O atom in the carboxyl group of LCA side chain formed a hydrogen bond with the hydroxyl group of T72,which could hold LCA in a catalytic pose to achieve high selectivity.In addition,by analyzing the hydrophobic interaction around LCA,it was found that the introduction of the hydrophobic amino acids(G87A,A74L and L181M)occupied the binding pocket and stabilized catalytic pose of LCA,resulting in enhancing the binding ability of LCA to catalytic center,which further explained why the mutant G87A/W72T/A74L/L181M could efficiently catalyze LCA to 1β-OH-LCA.