Study On Substrate Specificity And Stereoselectivity Of Ketone Reductase In Polyketone Synthesis

In recent years, chiral drugs play an increasingly important share of great value in the pharmaceutical market, and they attract the attention of scientists from various countries. Chiral drugs become a hot research in the world. The chiral alcohols and their derivatives are important precursors in the synthesis of chiral drugs. Compared with the conventional chemical reduction, Asymmetric reduction of Keto compound is an important method of chiral alcohols production, it showed lots of advantages, e.g. mild reaction conditions, good stereoselectivity, and envrionmental friendly, The most critical part for the enzymatic reduction is to find appropriate reductase.Polyketides produced by microorganism or plant are kinds of structural diversity of natural products, they are also important source of clinical medicine. Their biosynthesis involves the reduction of the keto-dimensional catalyzed by polyketide ketoreductase domain and modified bypost- polyketide synthase ketone reductase, respectively. This article studied several different KRs, including their polyketide biosynthetic pathway and their potentiality as biological catalyst in the synthesis of chiral alcohols. We identifie their substrate broadness and stereospecifity, and try to use the X-ray crystallography to analysis ketoreductase threedimensional structure to determine the catalytic centers in the substrate binding amino acid residues. The results of this study showed that:1、Three different polyketone synthase in KR structure domain(BacKR1, Tyl KR1 and EryKR1), reduction eight different substrates show that different KR has different substrate spectrum and stereo specificity. Three kinds of enzymes to the natural substrate analogues have catalytic activity. The difference is BacKR1 of cyclohexanone and 4- chloroacetophenone have some catalytic activity, Tyl KR1 have some catalytic activity of cyclohexanone, acetophenone, 4-chloroacetophenone, 4-fluoroacetophenone and ethyl acetoacetate, Ery KR1 have some catalytic activity of cyclohexanone, acetophenone and ethyl acetoacetate.Three kinds of enzymes in the reduction ability of ketone substrate have their respective advantages. Furthermore, the key amino acids site-directed mutagenesis on Tyl KR1 and Ery KR1 show that Tyl KR1 activity decreased, but have 2R, 3R configuration of the product; The configuration chang after EryKR1 mutation, not only produce 2S, 3R configuration also produce 2R, 3R configuration product. The site-directed mutagenesis dose affect the stereoselectivity of the enzyme, the mutation of key sites can also affect the enzyme activity.2、Use affinity chromatography and gel filtration chromatography to obtain four PKS KR Dnr U, DnrE, MeiF and Red1, use the method of NADPH consumption to study the substrate specificity. Finally find that MeiF and Dnr U can catalyze non-natural substrates, MeiF which have high catalytic activity, especially show higher catalytic activity to aromatic ketones, only MeiF in the four kinds of protein get protein crystals.