| KpADH is an NADPH-dependent alcohol dehydrogenase derived from Kluyveromyces polyspora for the efficient asymmetric synthesis of（4-chlorophenyl）-（pyridin-2-yl）-methanone（CPMK）reduction.In this study,the potential of KpADH in asymmetric reduction of sterically hindered biaryl ketone substrates was further explored.Wild-type KpADH（WT）was used as the starting enzyme and CPMK was used as the model substrate,and a series of substrates similar to CPMK were synthesized,as well as four heterocyclic-containing biaryl substrates.The substrate spectrum of CPMK was explored,and enantiocomplementary mutants were constructed by semi-rational engineering.Futhermore,the mechanism of stereoselective regulation was elucidated.The main research contents and results are as follows:First,in order to better understand the molecular mechanism by which KpADH recognizes diaryl substrates,we explored different biaryl substrates.The results showed that the activity and selectivity of the enzyme decreased as the para-chlorine atom changed to meta-and ortho-position,indicating that the para-chlorine group on the benzene ring played an important role in affecting the catalytic efficiency and stereoselectivity of KpADH.After changing the p-chloro group to p-trifluoromethyl（5a）and p-methyl（6a）,the enzyme selectivity was reversed to 49%（S）and 83%（S）,respectively.Four medicinal heterocyclic substrates were synthesized,namely thiophene ring,thiophene chloride ring,imidazole ring and cyclopentanedione,among which the activity and selectivity of the thiophene chloride ring substrate reached 10.8 U·mg-1and 81%（R）,further confirming that the para chlorine atom group plays an important role in the localization of the substrate inside the active center.Second,enantiocomplementary KpADH mutants were rationally designed to explore stereoselectivity laws.Site-directed mutagenesis of the substrate-binding pocket was performed to screen the sites involved in stereoselective regulation,and saturation mutations were performed on the key sites E214 and T215 to explore the distribution of amino acids.Further iterative mutagenesis successfully constructed the optimal mutants E214I/T215S/S237A（ISA）and F161V/S196G/E214G（VGG）,of which the（S）-type mutant ISA can catalyze the reduction of substrates 5a and 6a to generate biaryl alcohols.e.e.value>99%（S）,（R）-type mutant VGG can catalyze the reduction of 5a,6a to produce product alcohol with e.e.values of 99%（R）and98%（R）.Kinetic analysis showed that the KM values of mutant ISA and VGG for substrate 5a were improved compared with WT（0.44 m M）,which were 0.75 and 0.46 m M,respectively;for substrate 6a,mutant VGG was the same as WT（1.59 m M）,ISA decreased（0.77 m M）.In order to explore the molecular mechanism of protein crystallization and stereoselective regulation of KpADH,reveal the selective turnover mechanism of KpADH for substrates 5a and 6a,construct an inactive mutant Y164F and try to obtain the complex crystal structure of substrate,coenzyme and enzyme.The protein crystallization conditions were optimized,and high-quality crystals were finally obtained.15 sets of high-resolution crystal diffraction data were collected.However,no substrate was found in the active center after molecular replacement.Through homology modeling,the homology models of the enantio-complementary mutants ISA and VGG were obtained,and the molecular docking and interaction force analysis with the substrates CPMK,5a,and 6a were carried out,and the para-group of the biaryl ketone was preliminarily revealed.The steric hindrance of the substrate-binding pocket of KpADH and the important role of the alkyl-πinteraction in the regulation of stereoselectivity.In conclusion,this study expanded the application of KpADH in different biaryl substrate species,discovered the importance of para-substituents for stereoselectivity,rationally designed and constructed enantiocomplementary KpADH mutant enzymes,and based on molecular docking and kinetics.The analysis initially explored its chiral recognition mechanism. |
PDF Full Text Request