Among these enzymes, the phenylalanine ammonia lyase (PAL) (EC 4.3.1.5) and phenylalanine aminomutase (PAM) have been used for the synthesis of a broad range of arylalanines [9], [21], [29] and [31]. The industrial-scale production of PAL mainly utilizes the strains of the Rhodotorula genus [12] and [32]. We previously screened learn more strains
from soil and identified a Rhodotorula glutinis strain with higher PAL activity, which was denoted JN-1 (CCTCC M2011490). The full-length gene of the phenylalanine ammonia lyase (RgPAL) from R. glutinis JN-1was isolated and successfully expressed in E. coli [38]. The RgPAL is a member of the 4-methylene-imidazol-5-one (MIO)-dependent enzyme family, which includes PAL, histidine ammonia-lyase (HAL) [27], tyrosine ammonia-lyase (TAL) [20], and PAM and tyrosine aminomutases (TAM) [13], [14] and [29]. The MIO is a highly electrophilic prosthetic group that is formed post-translationally from a highly conserved Ala–Ser–Gly motif ( Fig. 1), which attacks the substrate
to facilitate the elimination of ammonia [24]. The RgPAL is shown to region-and-stereo selectively catalyze l-phenylalanine to trans-cinnamic acid and can be used to resolve dl-phenylalanine to produce the d-phenylalanine. The solubility of the trans-cinnamic acid is low at acidic side (about 0.006 g/L in aqueous solution at 25 °C), and the d-phenylalanine could be easily separated from the reaction solution through pH controlling. Therefore, the asymmetric resolution of racemic dl-phenylalanine by PAL is an attractive route and exhibits commercial application prospects. However, the optimum pH of Trichostatin A price RgPAL is 9 and the RgPAL exhibits low catalytic efficiency at acidic side; the trans-cinnamic acid exhibits high
solubility at pH 9 and the accumulated trans-cinnamic acid during the reaction inhibits the catalysis, which presents a significant barrier to RgPAL application. Therefore, a mutant RgPAL with a lower optimum pH is expected. The optimum pH of enzymatic activity is often determined by the ionizable Protein tyrosine phosphatase amino acids at active site that are involved in catalysis and substrate binding [30] and [36]. The key issue is that which ionizable amino acids can be accurately picked out, and the catalytic mechanism and structure analysis can provide useful information in this aspect [37]. The RgPAL acts through the Friedel–Crafts-type mechanism (Fig. S1) [1], [22] and [25]. In the reaction, the MIO attack the phenyl ring of the substrate to form carbocation 1 which would stabilize intermediate 2 formed by removal of the substrate’s C-3 hydrogen [1] and [3]. Collapse of the system to product occurs with the elimination of NH3 and the release of trans-cinnamic acid from the MIO. Reservations on this mechanism center on the potentially large energy barrier that must be surpassed in forming the carbocation intermediate [3].