Molecular Engineering Of ?-transaminase ArATA For Synthesis Of Chiral Naphthylethylamine

(R)-?+?-1-?1-naphthyl?ethylamine is a key chiral intermediate for the synthesis of calcimimetic drug cinacalcet hydrochloride.Biosynthesis of?R?-?+?-1-?1-naphthyl?ethylamine has attracted increasing attention.?-Transaminase has been considered to be potential for producing?R?-?+?-1-?1-naphthyl?ethylamine by asymmetric reduction of 1-acetonaphthone.Here,an?-transaminase ArATA from Arthrobacter sp.KNK168 was identified.ArATA could catalyze the asymmetric reduction of 1-acetonaphthone into?R?-?+?-1-?1-naphthyl?ethylamine with high stereoselectivity?99%,R?.Nevertheless,its catalytic activity is not sufficient for large-scale application at higher substrate concentrations.This study aims at engineering ArATA for improved catalytic activity.Here,ArATA was engineered by combinatorial strategies of random mutagenesis and semi-rational design.On one hand,a random mutation library was constructed by error-prone PCR.Meanwhile,a high-throughout screening method for transaminases suitable for 1-acetonaphthone as substrate was established and mutants with improved activities G136I,F225M,C281I,and T282S were identified.On the other hand,substrate docking analysis was performed based on the crystal structure of ArATA?PDB:3WWI?,and 16 residues were chosen.Among them,V69,W156,W192,G224,and A284 were found to be highly conserved.Therefore,alanine scannings were performed on remaining 11 residues,and G136,V199,and S223 were subjected to futher saturation and combinatorial mutagenesis.Variants V199W/S223P,G136I/V199W/S223P,and F225M/C281I were obtained with improved catalytic efficiency.The k_cat/K_m of variants V199W/S223P,G136I/V199W/S223P,and F225M/C281I showed9.9-,9.3-,and 4.4-fold of WT,respectively.Half-life of G136I/V199W/S223P was increased by 9%,indicating its improved thermostability.In substrate specificity analysis,V199W/S223P showed increased activity toward all tested ketones.The catalytic efficacy of variants V199W/S223P,G136I/V199W/S223P,and F225M/C281I were evaluated by asymmetric amination of 1-acetonaphthone?20 mM?for preparing optically pure?R?-?+?-1-?1-naphthyl?ethylamine?ee>99%?,in which 29.34%,16.17%,and 11.87%higher conversion rates were achieved by V199W/S223P,G136I/V199W/S223P,and F225M/C281I after 24 h,respectively.Finally,the conversion rates by variants V199W/S223P,G136I/V199W/S223P,and F225M/C281I were 81.78±0.14%,73.45±0.82%,and 70.74±3.30%,respectively.Based on molecular docking and molecular dynamics simulation,improved catalytic efficiency of V199W/S223P,G136I/V199W/S223P,and F225M/C281I could be attributed to its enhanced Pi-Pi T-shaped interaction with substrate 1-acetonaphthone,as well as increased interactions between more amino acid residues and the substrate.A slightly higher half-life of G136I/V199W/S223P was validated by its lower RMSF value of Loop 130?139 compared with WT.Additionally,molecular dynamics simulations in the pre-reaction state showed that the mutant V199W/S223P was more conducive to the reaction than WT.The results indicate that ArATA variants are promising biocatalysts in reductive amination of 1-acetonaphthone to achieve the efficient and enantioselective synthesis of?R?-?+?-1-?1-naphthyl?ethylamine.