Molecular Modification Of The Coenzyme-binding Domain Of Engineered Amine Dehydrogenases

NADH-dependent amine dehydrogenase(Am DH)-catalyzed asymmetric reductive amination of prochiral ketones and free ammonia has the advantages of mild reaction conditions,green efficiency and high optical purity of the product,and is widely used in important structural building blocks and pharmaceutical intermediates Synthesis of Chiral Amines.For Am DH whose substrate-binding domain and coenzyme-binding domain are independent of each other,the main researches in recent years have focused on the modification of key sites in the Am DH substrate-binding pocket,thereby improving the catalytic activity of Am DH substrates or broadening its substrate spectrum.However,less research has been done on the modification near the coenzyme binding domain.In this study,the molecular modification of the coenzyme binding domain of Am DH was carried out to obtain Am DH with high coenzyme affinity,which improved the catalytic efficiency of asymmetric reductive amination and thus reduced the cost of industrial production.The specific research contents of this paper are as follows:(1)By replacing the high coenzyme-affinity coenzyme-binding domain of leucine amine dehydrogenase(L-Bc Am DH)derived from Bacillus cereus for the coenzyme-binding domain of phenylalanine amine dehydrogenase(F-Bb Am DH)derived from Bacillus badius,the construction of A novel chimeric amine dehydrogenase c FLF-Am DH was obtained.The use of auto-induction medium enables soluble and high-efficiency expression of c FLF-Am DH.The coenzyme affinity(K_m)of c FLF-Am DH was 2-fold higher than that of F-Bb Am DH,and the activity for the natural substrate p-fluorophenylacetone(p FPA)was increased by 150%.The results of the enzymatic properties of c FLF-Am DH showed that the optimum reaction p H was 10.0 and the optimum reaction temperature was 60℃.Compared with the parental F-Bb Am DH,the thermal stability of c FLF-Am DH was improved by 160%.(2)Molecular docking studies showed that c FLF-Am DH has a shortened distance between residues at key sites where c FLF-Am DH acts on the substrate p FPA,which is the main reason for the improved catalytic activity.Molecular dynamics simulation(MD)and binding energy results showed that the fluctuation range of RMSD of c FLF-Am DH and NADH complex was smaller than that of F-Bb Am DH and NADH complex,and the RMSF value of c FLF-Am DH was lower than that of F-Bb Am DH,indicating that c FLF-The structure of Am DH is more stable than the parent.The results of asymmetric reductive amination showed that the conversion rate of c FLF-Am DH could reach 99%when the coenzyme concentration was 0.5 m M.(3)Based on sequence alignment with high coenzyme affinity leucineamine dehydrogenases(L-Bc Am DH and L-Es Am DH),amino acid residues(D155 and K193)near the coenzyme binding pocket were selected for polar amino acid saturation,respectively Mutation and combined mutation finally obtained a mutant strain F-Bb Am DH(D155L/K193N)with significantly improved coenzyme affinity and catalytic efficiency.The Km value of mutant F-Bb Am DH(D155L/K193N)for coenzyme NADH was 0.01 m M.(4)Molecular docking and molecular dynamics simulations of the complex between the enzyme and NADH showed that the substitution of the D155 site of F-Bb Am DH by leucine(L)affected the adjacent residues to indirectly alter the interaction between the enzyme and NADH,K193 The substitution of the site by asparagine(N)forms a tighter hydrogen bond with the pyrophosphate moiety of NADH.The results of asymmetric reductive amination showed that when the coenzyme concentration was 0.1 m M,the mutant F-Bb Am DH(D155L/K193N)could achieve 99%conversion within 1 h.To sum up,according to the characteristics of engineered Am DH having two independent domains,molecular modification of the coenzyme-binding domain of engineered Am DH can obtain mutants with significantly improved coenzyme affinity,thereby improving catalytic efficiency and reducing production costs.