화학공학소재연구정보센터
Enzyme and Microbial Technology, Vol.114, 22-28, 2018
One step synthesis of unnatural beta-arylalanines using mutant phenylalanine aminomutase from Taxus chinensis with high beta-regioselectivity
Phenylalanine aminomutase (TcPAM) from Taxus chinensis catalyzes the regioselective hydroamination of tram-cinnamic acid (t-CA) to yield beta-phe. However, the final product mixture consists of both alpha- and beta-phe owing t low regioselectivity, which is still a challenge to synthesize highly pure beta-phe. Therefore, a modified TcPAM wit high beta-selectivity is expected. Based on the catalytic mechanism and structure, two amino acid residues (Asn45 and Leu108) in active sites were identified as the key residues for controlling the regioselective hydroaminatio of t-CA and as promising candidates for mutagenesis to enhance beta-selectivity and decrease alpha-selectivity. Th Asn458 and Leu108 residues were mutated to yield variant TcPAM-Asn458Phe/Leu108Glu, and the beta-selectivity was approximately 5.2-fold higher than that of wild-type TcPAM, while alpha-selectivity decreased to 68%, and the percentage of beta-phe in the product mixture increased from 42% to 83%. In addition, the mutant was applied t synthesize beta-arylalanines using substituent t-CA as a substrate. The regioselectivity was also affected by the substituent groups at the phenyl ring of t-CA with respect to their electronic properties and position, and the 4-methoxy and methyl substituent t-CA were transferred into beta-arylalanines. The conversion rate also exceede 90%. In summary, the engineered TcPAM proved to be useful for one-step asymmetric amination of t-CA and it derivatives to synthesize highly pure beta-arylalanines.