Study In The Quantitative Structure-Activity Relationships And Reaction Kinetic Simulation Of Carbonyl Reductases

Carbonyl reductases are widely used for reducing prochiral carbonyl compounds into corresponding chiral alcohols that are widely used as intermediates for drugs and fine chemicals.The substrate spectrum of carbonyl reductases is one of the important evaluation indexes to characterize its catalytic performance and application value,the kinetics simulation of cascade reaction between carbonyl reductase and amine dehydrogenase can further study the reaction essential issues.In this article,we studied the structure-activity relationship between carbonyl reductase YtbE and its substrates by the method of 3D-QSAR(Three-Dimensional Quantitative Structure Activity Relationship),including the interactions between enzyme and substrate,the activity prediction and screening of new substrates from database.Double enzymes cascade reaction of carbonyl reductase and amine dehydrogenase for the conversion reaction of cyclohexanol to cyclohexylamine was studied by the kinetics simulation method.An aldo-keto reductase named YtbE was isolated and identified from Bacillus sp.ECU0013 based on the previous laboratory studies,which can catalyze an NADPH-dependent carbonyl reduction reaction with high stereoselectivity.3D-QSAR models were constructed to study the quantitative structure-activity relationship between enzyme and substrates.Two QSAR models of CoMFA(Comparative Molecular Field Analysis)and CoMSIA(Comparative Molecular Similarity Indices Analysis)were obtained,both the cross-validation coefficient(Q2)(0.623,0.601)are satisfactory,suggesting that the models are robust and effective for prediction.The detailed structure-function interaction relationships between the protein YtbE and different substrates were then examined by the contour maps analysis.It is proved that electrostatic and hydrophobic force fields play the most important role in determining the catalytic activity with contribution factors of 0.378 and 0.384 respectively.Furthermore,virtual screening using the ZINC15 database was performed to expand the substrate spectrum of YtbE.Based on the 77 potential catalytic substrates obtained from virtual screening,five were experimentally verified from the top 25 compounds,indicating a good accuracy of QSAR model.The 3D-QSAR assisted method was proved to be helpful for rational investigating and expanding of the YtbE substrate spectrum.In order to dig the application potential of carbonyl reductase deeply,the cascade reaction catalyzed by carbonyl reductase and amine dehydrogenase was studied.With the help of computer aided simulation method and experimental verification,the quantitative simulation model of the whole cascade reaction process was constructed to analyze the reaction process and optimize reaction conditions in silico.In this paper,the cascade reaction dynamics model was developed to study the most significant reaction process problems,such as inefficetnt coenzyme cycling,reversible reaction with low yield.The optimized enzyme concentrations,reaction rotating speed,and cosolvent ratio were obtained by the single-variable experiments.The addition concentration and proportion of oxidized and reduced coenzymes at 10 mM substrate concentration were optimized in silico for the maximum product yield through reaction kinetics model.Comparing to the wet experiment,we found that the fitting effect of the model is consistent with the trend of experimental results.The quantitative model could be further developed by the investigation on the operational stability of enzymes,which is expected to be widely used in the optimization of multi-enzyme reaction systems.