To successfully select those residues in the active site, a theoretical model of RgPAL was constructed through homology modeling using RtPAL (PDB ID: 1T6J) as the template. As shown in Fig. 2, all of the residues that were

the superimposed with RtPAL showed an RMSD of 0.224 Å ( Fig. 2A), and the Ramachandran plot suggests that 94.9%, 3.2%, and 1.9% of the residues in derived model are in acceptable region, marginal Selleck 17-AAG region and disallowed region, respectively ( Fig. 2B), These finding indicated that the model is reasonable and could be used in further molecular docking simulation. Using the AutoDock global–local evolutionary algorithm, we searched for those sites with the lowest free energy of binding between the ligand and the enzyme. As shown in Fig. 3, the active site cavity of RgPAL was bisected into

two regions ( Fig. 3A): one binds the amide group adjacent to the aromatic ring and the other binds the carboxyl group of the substrate. The phenyl ring of the substrate is roughly orthogonal to the plane of the MIO, and the methylidene of the MIO points to C2 of the aromatic ring ( Fig. 3A and B). In the carboxyl group binding pocket, the Arg361 residue is 3.2 Å from the carboxyl group of the substrate, and this residue might play a role in MK-1775 purchase the binding of the carboxylate moiety of the substrate through a salt bridge. The Tyr358 residue is 2.7 Å from the β-H of substrate, which is close enough to act as the β-H abstracted base ( Fig. 3C). The Glu491 residue is the closet residue to the amino group of the substrate (2.8 Å, Fig. 3C) and might accept the amino group of substrate as the enzyme base, which is consistent with the results reported by Bartsch [1]. The Tyr358, Arg361 and Glu491 are highly conserved in PAL ( Fig. 1). In the aromatic ring binding pocket, the His136 residue points to the phenyl ring of the substrate. The imidzaole group

of His136 is parallel to the phenyl ring and might generate a π–π interaction. Moreover, the imidazole of His136 and the adjacent amide group of Gln137 which points to the phenyl ring within a distance of 4.5 Å, form a hairpin motif to clamp the phenyl ring ( Fig. 3B and C). To verify the function of the hairpin, the His136, Gln137 were deleted (RgPAL-Δ136H, RgPAL-Δ137Q) and mutated to negative (RgPAL-H136E, triclocarban RgPAL-Q137E) and positive charges (RgPAL-H136K, RgPAL-Q137K) as well as uncharged amino acids (RgPAL-H136F, RgPAL-Q137L), respectively. The mutant and wild type RgPAL proteins appeared a single band of about 75 kDa on SDS-PAGE ( Fig. 4). The activities of RgPAL-Δ136H and RgPAL-Δ137Q were not detected (data not shown), suggesting that the residues at the two sites were essential for catalysis. The RgPAL-H136K, RgPAL-Q137K and RgPAL-H136E lost the enzymatic activity (data not shown), and the RgPAL-H136F, RgPAL-Q137L sharply decreased the activity ( Fig. 5). Compared with those mutants, the activity of RgPAL-Q137E decreased slightly ( Fig. 5).