Investigation into isomerization reaction of phenylalanine aminomutase from Pantoea agglomerans

摘要

Phenylalanine aminomutase (PaPAM) from Pantoea agglomerans is a member of the MIO (4-methylene-imidazol-5-one) family of enzymes, which isomerizes alpha-phenylalanine to beta-phenylalanine, and could be used to synthesize unnatural beta-arylalanine. However, the mechanism of isomerization reaction is not clear. To investigate the mechanism, the gene (pain), which encodes PaPAM, was first expressed in E.coli, and recombinant PaPAM was prepared using affinity chromatography. Then, N-15-(2S)-alpha-phenylalanine, (2S)-(3-H-2(2))-alpha-phenylalanine and (2S,3S)[2,3-H-2(2)]-alpha-phenylalanine were used as substrates to analyze the mechanism of isomerization reaction. The results of MS and NMR showed that the isomerization reaction was performed through the intramolecular exchange of NH2 with pro-3R hydrogen of alpha-phenylalanine. The PaPAM shuttles the alpha-NH2 of alpha-phenylalanine to beta site to replace the pro-3R hydrogen. Simultaneously, the pro-3R hydrogen is shifted to a site to produce beta-phenylalanine. Furthermore, a key residue, Phe at position 455 in the active site, was determined to control the exchange way using molecular docking and sequence alignment of MIO family enzymes. The results indicated that the key 455 Phe residue is involved in changing the binding orientation of the carboxyl group of the intermediate trans-cinnamic acid to control the NH2-H pair exchange.