Semi-rational Engineering Of Stereoselectivity Of A Diaryl-ketone Reductase And Its Crystallization

(R)-（4-Chlorophenyl）-（pyridin-2-yl）-methanol[（R）-CPMA]is a key chiral intermediate for the synthesis of Betahistine（antihistamine）.Enzymatic synthesis of chiral alcohols has becoming more preferrable owing to its advantages such as mild reaction conditions,high selectivity and conversion.In previous work,an NADPH-dependent short-chain alcohol dehydrogenase from Kluyveromyces polyspora（KpADH）was discovered by gene mining.It catalyzed the reduction of（4-chlorophenyl）-（pyridin-2-yl）-methanone（CPMK）to（R）-CPMA,with stereoselectivity of 82%e.e.In this thesis,semi-rational engineering of KpADH was performed to improve its stereoselectivity,and protein crystallization was carried out to further elucidate its stereo-recognition mechanism.The main research content is as follows:Semi-rational design was attempted to increase the enantioselectivity of KpADH.Based on the degeneracy of amino acid codons,a hydroclassified combinatorial saturation mutagenesis（HCSM）strategy was designed to construct a combinatorial saturation mutation library.The highest enantioselectivity of 99.4%e.e.（R）was obtained with 50C10,（C165F/E214Y/S237A）and its k_cat/K_m increased from 16.7 to 59.3 s^–1·mM^–1.50C10 showed increased specific activity and enantioselectivity toward almost all diaryl ketones tested,especially to 4-Br-benzophenone,4-NO₂-benzophenone,and bis-acetophenone,especially 4-Br-benzophenone,4-NO₂-benzophenone,and bis-acetophenone,in which over 99%e.e.was resulted.In asymmetric biosynthesis of（R）-CPMA catalyzed by 50C10,over 99%e.e.and 99%conversion were achieved in 12h at 500 mM CPMK（with substrate feeding）.HCSM library screening results revealed that 214 and 237 are key sites for stereoselective of KpADH.Effects of 214 and 237 residues on catalytic and stereoselectivity was studied by site-directed mutagenesis and combinational mutagenesis.After two rounds of mutations,mutant E214Y/S237A was obtained,capable of reducing CPMK to（R）-CPMA in 99.1%（R）e.e.Another mutant E214G/S237C with inverted stereoselectivity of 75.6%（S）was attained.In site-directed mutagenesis study,only single-point mutant E214G showed mild inverted stereoselectivity（6.71%（S））with respect to WT.These results demonstrate the importance of the combinatorial effect on inverted stereoselectivity of KpADH.The substrate affinity of mutants E214Y/S237A and E214G/S237C toward CPMK（0.62 mM and 0.31 mM）were were improved compared with WT（0.78 mM）.To further investigate and chiral recognition mechanism of KpADH,the crystal structure of KpADH-NADPH complex was resolved.High-quality protein crystals were obtained under conditions of 0.2 M ammonium acetate,0.1 M HEPES,pH 7.0,31%PEG 3,350,1 mM NADPH,and 5%isopropanol by using the sessile drop method.Crystal data resolution was collected by X-ray diffraction and strucrue of KpADH-NADPH complex with 1.98?was obtained by molecular replacement method.Based on the crystal structure and computational analysis,steric hindrance at the 214 and 237 is the main reason for the increased selectivity and catalytic activity of E214Y/S237A and E214G/S237C.In summary,our study provides an effective mutagenesis strategy to improve the enantioselectivity of KpADH,and crystal structure data of KpADH.