Mechanism-based site-directed mutagenesis to shift the optimum pH of the phenylalanine ammonia-lyase from JN-1.

Phenylalanine ammonia-lyase (PAL) from JN-1 stereoselectively catalyzes the conversion of the l-phenylalanine into -cinnamic acid and ammonia, and was used in chiral resolution of dl-phenylalanine to produce the d-phenylalanine under acidic condition. However, the optimum pH of PAL is 9 and the PAL exhibits low catalytic efficiency at acidic side. Therefore, a mutant PAL with a lower optimum pH is expected. Based on catalytic mechanism and structure analysis, we constructed a mutant PAL-Q137E by site-directed mutagenesis, and found that this mutant had an extended optimum pH 7-9 with activity of 1.8-fold higher than that of the wild type at pH 7. As revealed by Friedel-Crafts-type mechanism of PAL, the improvement of the PAL-Q137E might be due to the negative charge of Glu137 which could stabilize the intermediate transition states through electrostatic interaction. The PAL-Q137E mutant was used to resolve the racemic dl-phenylalanine, and the conversion rate and the value of d-phenylalanine using PAL-Q137E at pH 7 were increased by 29% and 48%, and achieved 93% and 86%, respectively. This work provides an effective strategy to shift the optimum pH which is favorable to further applications of PAL.