Probing the Catalytic Center of Porcine Aminoacylase 1 by Site-Directed Mutagenesis, Homology Modeling and Substrate Docking

Abstract

Three-dimensional structural models of porcine aminoacylase 1 (pACY1) were constructed by homology modeling and aligning the structures of members of the M20 peptidase family. After energy minimization and quality evaluation, the best model from the homology modeling was chosen for docking with the best substrate (N-acetyl-L-methionine). The most reasonable binding mode was found among a large number of predicted complexes by using clustering analysis and screening with expert knowledge. Structural analysis revealed that the zinc ion is not likely to bind to the substrate, and that Arg348 and Glu146 play vital roles in binding and catalysis. In the site-directed mutagenesis experiments, mutation of His79, Asp112, Glu147, Arg348, and Glu146, resulted in significant reductions of specific activity, while the wild-type pACY1 overexpressed in Rosetta (DE3) had almost as high a specific activity as the native enzyme. On the basis of these observations, we proposed a revised catalytic mechanism for this metalloenzyme.

Characterization of thermostable aminoacylase from hyperthermophilic archaeon Pyrococcus horikoshii

“…5A) was very similar to the structure of Bsb AH (PDB ID: 1YSJ), which has 39% homology to Pho ACY and two atoms of nickel in one subunit, and that the spatial configuration of the amino acid residues related to zinc binding (Fig. 5B) also resembled that of Bsb AH, pACY1 [17] and the zinc‐binding domain of the T347G mutant from hACY1 (PDB ID: 1Q7L) [23], although His164 was replaced by a glutamic acid residue in pACY1 and hACY1. In this model, the residues His106, Glu139, Glu140 and His164 were located close to each other and formed a metal‐binding pocket, as found in the other aminoacylases, leading to the conclusion that these four residues are zinc ligands.…”

“…The active site of Pse CP contains two zinc atoms bridged by a water molecule (which acts as an attacking hydroxyl ion nucleophile), together with a glutamic acid residue (Glu175 of Pse CP) (which plays a role as a general base in hydrolytic catalysis) [25]. The hydrolytic catalysis of l ‐aminoacylases, including Pho ACY, seems to obey a mechanism similar to that proposed in pACY1, where the water molecule binding the zinc metal and glutamic acid residue act as a nucleophile and general base, respectively [17].…”

Increasing the synthesis/hydrolysis ratio of aminoacylase 1 by site-directed mutagenesis

“…In order to map electrostatic contributions in the active site of pAcy1 that determine the pKa of E146, we constructed a homology model for the catalytic domain of the enzyme based on a published procedure [27] with additional validation of structural integrity. Individual quality scores for residues within 8 Å of the catalytic base supported the accuracy of our model.…”

“…The pAcy1 homology model was build with the program MODELLER 9V4 (http://salilab.org/modeller/) as described previously by Liu et al [27] with slight modifications and with additional analysis of topological errors and energy minimization as described in Supplementary material.…”

Aminoacylase I deficiency due to ACY1 mRNA exon skipping

“…The p.Lys308Glufs*7 ACY1 amino acid sequence was used as target for the HM analysis and the structure of CPG2 (15) [chains A and D of protein database (PDB) entry 1CG2] as template, as described previously for porcine ACY1 (16). The p.Lys308Glufs*7 ACY1 amino acid sequence was used as target for the HM analysis and the structure of CPG2 (15) [chains A and D of protein database (PDB) entry 1CG2] as template, as described previously for porcine ACY1 (16).…”