Study On Enantioselectivity And Catalytic Promiscuity Of Reductase

Sitagliptin is a new hypoglycemic drug developed by Merck in the United States for the treatment of type 2 diabetes.3-hydroxy-4-?2,4,5-trifluorophenyl?butanoate methyl ester[?S?-HTB]is key intermediate in the catalytic synthesis of Sitagliptin.In this paper,the recombinant whole cells of aldo-keto reductase?aldo-ketoreductases,AKR?and glucose dehydrogenase?glucosedehydrogenase,GDH?were screened for asymmetric reduction of substrate3-oxo-4-?2,4,5-trifluorophenyl?butanoate methyl ester?OTB?.three kinds of AKR-GDH recombinant whole cells showed different catalytic performances in the process of asymmetric synthesis of HTB.When ytbE-GDH whole-cell catalytic substrate OTB mainly produces?R?-HTB,whileiolS-GDH and yhdN-GDH whole-cell catalytic OTB,the product HTB was mainly?S?-HTB.yhdN-GDHrecombinantwhole cell,which showed the best catalytic performance in the process of asymmetric synthesis of?S?-HTB,was selected to continue the study.The reaction conditions involved in the asymmetric synthesis of?S?-HTB catalyzed by yhdN-GDH whole cell were optimized.the conversion rate and enantiomeric excess value?e.e._p?of?S?-HTB synthesis by whole-cell yhdN-GDH under the optimum conditions were 24.9%and 85.8%,respectively.In order to further improve the conversion rate of yhdN-GDH recombinant whole-cell catalytic synthesis of?S?-HTB and the enantiomeric excess value of the product.The ligand molecule OTB was docked with yhdN.Through the docking results,a series of mutants of yhdN and GDH recombinant whole-cell were selected tocatalyzesubstrateOTB.Among them,the yhdN_L278G-GDH recombinant whole cell increased the reactionconversion and the enantiomeric excess value?e.e._p?of the product to 33.8%and 92.0%without additional coenzyme.The reason for this phenomenon is that when the leucine at position 278 of yhdN is mutated to glycine,the volume of amino acid residues changes from large to small,and the protein changes in space,which leads to the change of the results of the reaction.Next,the docking results of ligand molecule OTB with yhdN and yhdN_L278G were compared.,It is found that compared with yhdN,yhdN_L278G has two new interactions with the substrate.One is that the O atom of the 323th methionine residue?Met323?interacted with the C atom at the end of the straight chain of the substrate,The other is the interaction between the O atom of the aspartic acid residue?Asp52?at the active site52 ofyhdNand the F atom connected to the C atom of the benzene ring.From the molecular structure of the substrate,it can be analyzed that the increased two kinds of interactions are located at both ends of the substrate molecules,the increased two kinds of interactions and the five kinds of interactions before mutation,which can better stabilize the position of the substrate after entering the active pocket.As a result,the conversion of the reaction and the enantioselectivity of aldo-keto reductase were improved.Organic halogen compounds have become serious environmental pollutants,but many physical methods for the treatment of organic halogen compounds will cause secondary pollution to the environment,so the biological treatment of halides has attracted more and more attention.In this paper,ene-reductase was chosen to catalyze dehalogenation.In this paper,ene-reductase was chosen to catalyze dehalogenation,Theene-reductase could be bypurified by Ni-NTA metal-affinity chromatography.ER pureenzymecatalyzed3,4'-Dichloropropiophenonesynthesis4'-chlorophenylacetone.Then,the conditions involved in the catalytic reaction were optimized,and the conversion of the reaction was 49.5%under the optimum conditions.Under the optimized conditions,ER was used to catalyze 1-?4-chloro-phenyl?propenone,and it was found that 4'-chlorophenylacetone could not be formed.This negates the catalytic mechanism that the dehalogenation reaction substrate is first spontaneously dehydrohalogenated and then“C=C”was reduced to“C—C”by ene-reductase.The catalytic promiscuity of ene-reductase was verified.The dehalogenation reaction catalyzed by ER has been reported before,but the kinds of catalytic substrates are esters and lactones.In this paper,the ene-reductase with catalytic promiscuity was successfully used to catalyze the dehalogenation of halogenated ketones as non-natural substrates.The main work of this paper focuses on oxidoreductase.The enantioselectivity of aldo-keto reductase and the catalytic promiscuity of ene-reductase were studied.UsedAKR-GDH whole cells reduce OTB to produce?S?-HTB with low cost and mild reaction conditions.The ene-reductase was used to synthesis4'-chlorophenylacetonefrom 3,4'-Dichloropropiophenone.It further expands the application of oxidoreductase in the field of drug synthesis and environmental protection.