Engineering An Amine Dehydrogenase For Reductive Amination Of Bulky Aromatic Ketones

Chiral amines are a class of important building blocks for the synthesis of many bioactive molecules such as drugs and natural products.They are also used as chiral resolving agents and chiral ligands to produce various fine chemicals and agricultural chemicals.Amine dehydrogenase(AmDH)is a new type of biocatalyst developed in recent years for catalyzing the asymmetric reductive amination of ketones to chiral amines.It can use inexpensive ammonia as an amino donor,and the by-product is only water.It has good atomic economy and shows good application prospects in the synthesis of chiral amines.However,the amine dehydrogenases reported so far are not able to catalyze the reductive amination of bulky aromatic ketones.Herein,we intend to engineer an amine dehydrogenase Gk-AmDH to achieve a breakthrough in the catalytic activity towards bulky aromatic ketone compounds,extend its substrate scope and provide a potential biocatalyst for the synthesis of bulky aromatic chiral amines.The main work are the following two parts:Part Ⅰ:In view of the current difficulty that AmDH cannot accept bulky aromatic ketones.we used the amine dehydrogenase Gk-AmDH constructed before as the template for further research,and carried out homo-modeling and structural analysis via bioinformatic methods.Using bulky benzylacetone as the model compound,and molecular docking with Gk-AmDH was conducted subsequently.Based on the docking results,we speculated that the amino acid residues at the end of the substrate-binding pocket(L50,V144,V309 and A310)may have the steric hindrance effect on the binding of the substrate.Therefore,we mutated them to smaller alanine or glycine,and examined the effect of these mutations on enzyme catalysis.On this basis,we combined three beneficial mutations and finally obtained the mutant Gk-AmDH-M3,which has a 10-fold increase in catalytic efficiency towards the model compound benzylacetone,with activity of 0.65 U mg-1,much higher than the highest value(8.8 mU mg-1)reported in the literature before.Subsequently,we measured the melting temperature of the best mutant Gk-AmDH-M3,and its Tm value is 68.6℃.showing excellent thermal stability.In addition,by measuring the volume of the substrate-binding pocket,we found that the pocket volume of the best mutant was expanded by 44 (?)3 compared to that of the starting enzyme,showing the potential of binding many other bulky substrates.Part Ⅱ:After obtaining the mutant Gk-AmDH-M3 with a maxmium increase in the volume of the substrate-binding pocket,we investigated its catalytic ability for various bulky aromatic ketones to explore its substrate scope.The result shows that the starting enzyme cannot catalyze most of the bulky aromatic ketone substrates,but the best mutant Gk-AmDH-M3 can transform them easily.In addition,based on the reaction results and structural analysis,we found that the S156 site may interact with the ortho-substitution group in the aromatic ring of the substrate,which affects the catalytic efficiency of such compounds.Therefore,we conducted a site-directed saturation mutation on it and found that there is a hydrogen bonding interaction between this site and the ortho-substitution group in the aromatic ring of the substrate,which provides a good basis for the subsequent research of AmDH.Finally,after verifying that the mutant Gk-AmDH-M3 has good catalytic capacity for various bulky aromatic ketones,we constructed the coenzyme recycle system by coupling formate dehydrogenase(FDH)with ammonium formate for synthesizing the bulky chiral amine products.The by-products of the process were only water and carbon dioxide.The results show that the product conducted by enzymatic synthesis was R-selectivity and optical purity of them show above 99%(>99%ee).The abovementioned result comfirmed the feasibility of amine dehydrogenase in the synthesis of bulky aromatic chiral amines.