Substrate specificity of the phenolic acid decarboxylase from Lactobacillus plantarum and related bacteria analyzed by molecular dynamics and docking

Improving the catalytic characteristics of phenolic acid decarboxylase from Bacillus amyloliquefaciens by the engineering of N-terminus and C-terminus

“…P-m1 mutation from the His92 to Glu92 resulted in a reduction of the k m for four phenolic acids from 4.09 mmol/L, 2.39 mmol/L, 3.10 mmol/L, 3.20 mmol/L to 1.88 mmol/L, 2.08 mmol/L, 1.36 mmol/L, 1.82 mmol/L, as well as for the mutation from the Tyr122 to Leu122 resulted in the alteration of the K m for four substrates to 1.22 mmol/L, 1.00 mmol/L, 6.49 mmol/L, 4.55 mmol/L, whereas the k cat / K m of these four increased by 56, 74, 229, 113% respectively. The docking data of a previous study suggested that the changes involving the residues with different charges may affect the orientation of the matrix in the cavity before entering the active site [ 17 ]. In comparison with P-WT, the affinity of P-m1 to four substrates as well as P-m2 to the PCA and the FA showed a great improvement, however, the affinity of P-m2 to the CA and SA decreased.…”

“…The entry region was reported to be comprised of the amino acid side chains Asn22 (15) (original P-WT numbering in bracket), Trp24 (17), Met44 (37), Glu99 (92), Pro102 (95), Leu129 (122), Val131 (124), and Glu133 (126) [ 9 ]. Sequence comparison of a large number of active sites identified that a certain degree of variation was shown by some sites in the substrate-binding cavity, especially E99 (92), L129 (122), and V131 (124), which were closely related to the combination of the enzyme and the substrate and the catalytic mechanism [ 17 ].…”

“…These residues played an important role in the substrate specificity of PAD. A detailed analysis of the molecular interaction between the substrate binding cavity and trans-p-coumaric acid in these structures shows that the substrate can be stabilized by two hydrogen bonds with residues E99 and W24 when E–L–V is present [ 17 ]. So we created six hybrid enzyme by replacing the C-terminal extension (P-C) (the name of the mutant in bracket) and N-terminal extension (P-N) of P-WT with that of chain A of Lactobacillus plantarum (PDB code: 2W2A) [ 9 ], and site-directed mutagenesis of His92Glu (P-m1), Tyr122Leu (P-m2) and combination the N-terminal replacement with His92Glu (P-Nm1), Tyr(122) Leu (P-Nm2).…”

Improving the catalytic characteristics of phenolic acid decarboxylase from Bacillus amyloliquefaciens by the engineering of N-terminus and C-terminus

“…Besides, it was reported that there is a common cavity or gap between the two pieces of each subunit [16] and that the cavity becomes larger near the molecular surface and gradually narrows as the enzyme enters. The entry region was reported to be comprised of the amino acid side chains Asn22 (15) (original P-WT numbering in bracket), Trp24 (17), Met44 (37), Glu99 (92), Pro102 (95), Leu129 (122), Val131 (124), and Glu133 (126) [9]. Sequence comparison of a large number of active sites identified that a certain degree of variation was shown by some sites in the substrate-binding cavity, especially E99 (92), L129 (122), and V131 (124), which were closely related to the combination of the enzyme and the substrate and the catalytic mechanism [17].…”

“…These residues play an important role in the substrate specificity of PAD. A detailed analysis of the molecular interaction between the substrate binding cavity and trans-p-coumaric acid in these structures shows that the substrate can be stabilized by two hydrogen bonds with residues E99 and W24 when E-L-V is present [17]. So we created six hybrid enzyme by replacing the C-terminal extension (P-C) (the name of the mutant in bracket) and N-terminal extension (P-N) of P-WT with that of chain A of Lactobacillus plantarum (PDB code: 2W2A) [9].…”