Probing the Acyl-Binding Pocket of Aminoacylase-1

Abstract

The aminoacylase-1/metallopeptidase 20 (Acy1/M20) family features several l-aminoacylases useful in biocatalysis. Mammalian Acy1, in particular, has been applied in racemic resolution and reverse hydrolysis. Despite recent advances in our understanding of the active site architecture and functioning, determinants of Acy1 substrate specificity have remained uncharted. Comparison to bacterial homologues points to a sterically more restricted acyl-binding pocket for Acy1. Here we sought to map characteristics of the acyl-binding pocket of human and porcine Acy1. Toward this end, we determined Michaelis constants for an analogue series of aliphatic N-acyl- l-methionine substrates and translated the values into three-dimensional quantitative structure-activity relationship models employing the minimal topological difference-partial least square method. The QSAR models for the two enzymes suggest overall similar binding pockets in the acetyl-binding portion and indicate a general preference for straight-chain acyl moieties. Embedding of the QSAR map for human Acy1 in the structure of its metal-binding domain associates the side chain of Ile177 with limited acyl chain elongation which was not observed for the porcine enzyme. The topological model further supports roles of Thr347 and Leu372, which are both conserved in the porcine enzyme, in restricting acyl chain branching at the alpha- and beta-positions, respectively. Mutational analyses confirmed our predictions for Thr347 and Leu372. Moreover, the T347S variant of human Acy1 exhibited markedly increased catalytic efficiency against N-benzoylamino acids, demonstrating the potential for engineering of substrate specificity in Acy1. We discuss the more general application of the employed procedure for protein design.

Fast, Continuous, and High-Throughput (Bio)Chemical Activity Assay forN-Acyl-l-Homoserine Lactone Quorum-Quenching Enzymes

“…This AH was anticipated to be at least an acceptable substrate for both aminoacylases with regard to its amino acid and acyl moieties (35,36) and was obtained by heat-accelerated autohydrolysis of C 6 -HSL (see Fig. S3 in the supplemental material).…”

“…Probably other unspecific protein purification methods like hydrophobic interaction or ion-exchange chromatography would be suitable as well. If the specific activity of pAcy1 is too low toward a certain type of AH, most likely those with very bulky or branched acyl side chains (35), and this cannot be overcome by higher enzyme concentrations, one should consider the use of pAcy1 variants (35) or even other aminoacylases instead. Moreover, cells that should be examined for QQ enzymes have to be washed with a buffer solution prior to cell disruption.…”

Synthesis and evaluation of 18F labeled FET prodrugs for tumor imaging

“…One subclass features degradative dipeptidases and exopeptidases (EC 3.4), while the other comprises of various amidohydrolases acting on carbon–nitrogen bonds rather than peptide bonds (EC 3.5) (2, 3). [35, 36]. Mammalian Acy1, in particular, has been applied in racemic resolution and reverse hydrolysis.…”

Fully automatized high‐throughput enzyme library screening using a robotic platform

“…For this, mutations in the acyl-binding pocket of porcine aminoacylase 1 (pAcy1) were combined to identify more active variants towards N-benzoyl-L-methionine (NBM). Lindner et al (2008) predicted that the residues T345, L370 and to a minor degree L176 restrict the acyl-binding pocket of pAcy1 and therefore the conversion of N-acyl-L-amino acids with bulky acyl groups. Here, T345G served as basis for a mutant library (L176X, T345G, L370X) as the variant T347G in the highly homologous human aminoacylase 1 showed much higher activity towards NBM than the wild type (Lindner et al, 2008).…”

“…1 and 2). The suitability and general applicability of this platform is exemplified for four different enzymes with relevance for biocatalysis: a (S)-selective amine transaminase [VFL-ATA, from Vibrio fluvialis (Genz et al, 2015)], a Baeyer-Villiger monooxygenase [CHMO, cyclohexanone monooxygenase from Acinetobacter calcoaceticus (Donoghue et al, 1976)], two haloalkane dehalogenases [(HLD, DhaA from Rhodococcus rhodochrous (Newman et al, 1999), and DhlA from Xanthobacter autotrophicus (Janssen et al, 1989)] and porcine acylase I [(pAcyl, from Sus scrofa (Lindner et al, 2008; for details see Table SI, Supporting Information)]. In all four cases, mutant libraries were created and screened for variants with altered properties using the automated robotic platform (Figs.…”