Modulating Stereoselectivity Of Diaryl Alcohol Dehydrogenase:Engineering And Mechanism Studies

Optically active diaryl alcohols is an important class of chemical chiral compounds which could be applied in the synthesis of pharmaceuticals agrochemicals and natural products.Among them,?S?-?4-chlorophenyl?-?pyridine-2-yl?-methanol[?S?-CPMA)]is a vital intermediate for anti-allergic drug Bepotastine and could be synthesized by asymmetric reduction of its corresponding?4-chlorophenyl?-?pyridine-2-yl?-ketone?CPMK?.Due to the strong steric hindrance of the diaryl groups,very few alcohol dehydrogenases have been reported with desirable activity and enantioselectivity.Previously,an NADPH-dependent short-chain alcohol dehydrogenase from Kluyveromyces polyspora?KpADH?has been identified as a robust biocatalyst due to its high conversion of diaryl ketone substrate?4-chlorophenyl?-?pyridine-2-yl?-ketone?CPMK?with a moderate R-selectivity of 82%ee.In this study,we focused on the research of Kp ADH to obtain biocatalyst with excellent enantioselectivity by molecular engineering.Additionally,the studies on stereoselective catalytic mechanism of diaryl alcohol dehydrogenase as well as?S?-CPMA continuous preparation were carried out.The main conclusions are as follows:?1?We have developed a carbonyl group-dependent colorimetric method in assay of carbonyl reductases using inexpensive 2,4-dinitrophenylhydrazine?DNPH?.This DNPH method has high sensitivity and low background disturbance,and can be used for high-throughput screening of carbonyl reductases toward various ketones including diaromaticketones,aliphaticketones/diketones,andaromaticketones.For1-?4'-clorophenyl?-1-?pyridine-2'-yl?-methyl ketone?CMPK?,a characteristic absorbance peak at around 500 nm was observed for the product of CPMK and DNPH with a molar absorbance coefficient of 13750 L·mol^-1·cm^-1.Significantly,this method is also amendable to whole-cell system for facile high-throughput screening and substrate specificity profiling.In random mutagenesis of a diaromatic ketone reductase KpADH,three mutants?M131F,S196Y and S237A?with improved activity toward CMPK were identified using DNPH method.The specific activities of M131F,S196Y and S237A toward CPMK are 1.5,1.3 and 1.8 folds of WT Kp ADH respectively.Additionally,the ee of?R?-CPMA produced by S237A was increased to 96.1%?R?while the mutant S196Y exhibited decreased enantioselectivity?74.7%,R?,indicating the evolvability of enantioselectivity.?2?The homology model of KpADH was constructed based on the crystal structure of Saccharomyces cerevisiae NADPH-dependent methylglyoxal reductase GRE2?PDB:4PVC??EC 1.1.1.283?.After eliminating the catalytic triad?S126-Y164-K168?,sixteen residues were selected for further mutagenesis study,they were subjected to“polarity scanning”by single mutation.Asparagine and proline were selected as"polar sieves"and"non-polar sieves",respectively.At last,six residues of Q136,F161,S196,E214,T215,and S237 were obtained and saturation mutagenesis?SM?of these six residues was performed using the NNK codon.After that,the iterative combinatorial mutagenesis?ICM?libraries were constructed using a data-based"Progressive Optimal Combination"?dPOC?approach.After iterative combinatorial mutagenesis,variants Mu-R2 and Mu-S5 with enhanced?99.2%ee,R?and inverted?97.8%ee,S?stereoselectivity were obtained.?3?To probe the molecular mechanism of stereoselective inversion,we crystallized WT KpADH,Mu-R2 and Mu-S5 of.and refined to resolutions of 1.98,2.20,and 1.78?.based on MD simulation,Mu-R2-CPMK_ProR and Mu-S5-CPMK_ProS were more favorable in the formation of prereaction states when two distances of d(O14_CPMK-H9_Y164)?3.4?and d(C7_CPMK-H4_NPH)?4.5?were used to represent the prereaction states.Interestingly,a quadrilateral plane formed by?-carbons of four residues?N136,V161,C237,and G214?was identified at the entrance of the substrate binding pocket of Mu-S5;this plane acts as a“polar gate”for substrates.Due to the discrepancy in charge characteristics between chlorophenyl and pyridine substituents,the pro-S orientation of CPMK is defined when it passes through the“polar gate”in Mu-S5,whereas the similar plane in wild type is blocked by several aromatic residues.?4?A robust two-enzyme system composed of immobilized Mu-S5 and BmGDH was developed via immobilization on Ni-NTA agarose for the stereoselective production of?S?-CPMA.The immobilized ketoreductase/glucose dehydrogenase system was continuously used in a flow reactor for a week at a flow rate of 5 mL·min^-1,a substrate concentration of 10mM,anoptimumenzymeactivityrationof2:1?Mu-S5:BmGDH?and hydroxypropyl-?-cyclodextrin as cosolvent.The substrate conversion rate can be maintained at 100%within first 3 days and giving space-time yield of 1560 g·L^-1·d^-1.The half-lif period of the immobilized two-enzyme system was approximate to 7 days.Finally,the chiral diaryl alcohol?S?-CPMA was absorbed by D101 resin and giving 84%isolated yield.