One step synthesis of unnatural β-arylalanines using mutant phenylalanine aminomutase from Taxus chinensis with high β-regioselectivity

Abstract

Phenylalanine aminomutase (TcPAM) from Taxus chinensis catalyzes the regioselective hydroamination of trans-cinnamic acid (t-CA) to yield β-phe. However, the final product mixture consists of both α- and β-phe owing to low regioselectivity, which is still a challenge to synthesize highly pure β-phe. Therefore, a modified TcPAM with high β-selectivity is expected. Based on the catalytic mechanism and structure, two amino acid residues (Asn458 and Leu108) in active sites were identified as the key residues for controlling the regioselective hydroamination of t-CA and as promising candidates for mutagenesis to enhance β-selectivity and decrease α-selectivity. The Asn458 and Leu108 residues were mutated to yield variant TcPAM-Asn458Phe/Leu108Glu, and the β-selectivity was approximately 5.2-fold higher than that of wild-type TcPAM, while α-selectivity decreased to 68%, and the percentage of β-phe in the product mixture increased from 42% to 83%. In addition, the mutant was applied to synthesize β-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 β-arylalanines. The conversion rate also exceeded 90%. In summary, the engineered TcPAM proved to be useful for one-step asymmetric amination of t-CA and its derivatives to synthesize highly pure β-arylalanines.

Keywords: Phenylalanine aminomutase; Site-directed mutagenesis; β-Arylalanine; β-Regioselectivity.