Genome Data Mining Of Quinuclidinone Reductase And Investigation On Molecular Catalytic Mechanism

Optically active（R）-3-quinuclidinol,a bicyclic chiral secondary alcohol linked by nitrogen,is an important chiral building block for many pharmaceuticals.（R）-3-quinuclidinol can be used to synthesize anticholinergic drugs,such as aclidinium bromide and revatropate for the treatment of chronic obstructive pulmonary disease（COPD）and talsaclidine for the treatment of Alzheimer’s disease（AD）.The purpose of this study was to discover highly efficient and stereoselective ketoreductases for cost-effective and efficient preparation of（R）-3-quinuclidinol,and to perform molecular engineering for finely manipulating the enantioselectivity.The main work and results are as follows:（1）Gene mining and screening of ketoreductases.After evaluation the activity of candidate probes,Ar QR was selected as the probe for gene mining.Twenty-one putative ketoreductases were heterogeneously expressed and submitted for functional screening.The specific activity of Ks KR and Ka KR were 1.3 times and 1.9 times of Ar QR,respectively,and all displayed enantioselectivity of>99%（R）.（2）Characterization of enzyme properties and optimization of conditions for synthesis of（R）-3-quinuclidinol.Purified Ks KR and Ka KR were obtained through nickel affinity chromatography.Ks KR and Ka KR were NADH-dependent with specific activities were 100U·mg^-1 and 110 U·mg^-1,respectively.Ks KR showed the highest activity at p H7.0 and 60°C,the half-life of 62.6 h at 30°C;Ka KR exhibited the highest activity at p H7.0 and 55°C,and half-life of 47.8 h at 30°C.EDTA has no obvious effect,and none metal ions could significantly enhance their activity.Kinetic parameter analysis revealed that the K_M and k_cat toward 3-quinuclidinone of Ks KR were 1.02 m M and 89.4 s^-1;Ka KR displayed higher binding affinity toward 3-quinuclidinone,with K_M of 0.8 m M and k_cat of 66.2 s^-1,and also high affinity toward NADH with K_M of 0.04 m M.Based on the above analysis,Ka KR was selected for asymmetric reduction.First,glucose dehydrogenase,formate dehydrogenase,and alcohol dehydrogenase were introduced to construct three enzyme-coupled regeneration system,to reduce the cost of cofactor.Ka KR-Bm GDH system can completely convert 0.5 M 3-quinuclidinone to（R）-3-quinuclidinol within 2.5 h,ranking the highest efficient.Then,the ratios of two enzymes were optimized,and the maximum catalytic efficiency was obtained at ratio of 1:2.5 of Ka KR and Bm GDH.Finally,the substrate concentration was increased without external aid of NAD⁺,and648 g·L^–1 of 3-quinuclidone could be completely converted into（R）-3-quinuclidinol at 7.5 h,with ee value of>99%,which is the highest substrate concentration that can be achieved without additional cofactors.（3）Molecular catalytic mechanism analysis of Ka KR and inversion of enantioselectivity.The alanine scanning of 24 residues lining the substrate binding pocket of Ka KR revealed that the key residues affecting substrate specificity and stereoselectivity were V94,S95,M194 and E198.Mutant V94A could increase the enzyme activity by 1.94 times,and the other three mutants were inactivated after being mutated to alanine.These four sites were studied in depth,and further mutated into amino acids with similar or opposite side-chain properties,and the kinetic parameters of the mutants were determined.The activity of V94G was 84.4 U·mg^-1,higher than WT and V94A indicating that there is steric hindrance at V94.There is electrostatic interaction between E198 and the N atom of 3-quinuclidinone,which could strictly fix the orientation of substrate to produce（R）-3-quinuclidinol.Mutant E198R with 92%（S）was obtained after semi-saturation mutagenesis at E198,and the double mutant E198R/F189K and E198F/F189K were obtained after combinatorial mutation with other enantioselectivity related sites,with enantioselectivity of 99%（S）.The Ka KR and double mutant E198R/F189K obtained in this study provide efficient catalysts for the synthesis of（R）-3-quinuclidinol and（S）-3-quinuclidinol,demonstrating its great potential in scaled-up manufacturing of optically active N-heterocyclic secondary alcohol.