Different roles for the acyl chain and the amine leaving group in the substrate selectivity of N-Acylethanolamine acid amidase

Andrea Ghidini,Laura Scalvini,Francesca Palese,Alessio Lodola,Marco Mor,Daniele Piomelli

doi:10.1080/14756366.2021.1912035

Andrea Ghidini, Laura Scalvini + Show 4 more

Open Access

https://doi.org/10.1080/14756366.2021.1912035

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Abstract

N-acylethanolamine acid amidase (NAAA) is an N-terminal nucleophile (Ntn) hydrolase that catalyses the intracellular deactivation of the endogenous analgesic and anti-inflammatory agent palmitoylethanolamide (PEA). NAAA inhibitors counteract this process and exert marked therapeutic effects in animal models of pain, inflammation and neurodegeneration. While it is known that NAAA preferentially hydrolyses saturated fatty acid ethanolamides (FAEs), a detailed profile of the relationship between catalytic efficiency and fatty acid-chain length is still lacking. In this report, we combined enzymatic and molecular modelling approaches to determine the effects of acyl chain and polar head modifications on substrate recognition and hydrolysis by NAAA. The results show that, in both saturated and monounsaturated FAEs, the catalytic efficiency is strictly dependent upon fatty acyl chain length, whereas there is a wider tolerance for modifications of the polar heads. This relationship reflects the relative stability of enzyme-substrate complexes in molecular dynamics simulations.

Highlights

N-acylethanolamine acid amidase (NAAA) is a cysteine hydrolase that catalyses the intracellular degradation of the lipid-derived messenger, palmitoylethanolamide (PEA), to palmitic acid and ethanolamine[1,2]
These include saturated fatty acid ethanolamides (FAEs) species with chain length varying from C13 to C18 (Figure 1A), monounsaturated species with chain length varying from C15 to C20 and a double bond in Z configuration located at the D9 or D10 position (Figure 1(B))
Our results show the existence of a strict correlation between the catalytic efficiency of the enzyme and the length of its substrate acyl chain, with an optimum for PEA

Summary

Introduction

N-acylethanolamine acid amidase (NAAA) is a cysteine hydrolase that catalyses the intracellular degradation of the lipid-derived messenger, palmitoylethanolamide (PEA), to palmitic acid and ethanolamine[1,2]. The X-ray structure of the rabbit enzyme in complex with a molecule of myristic acid strongly suggests that PEA places its 16-carbon saturated chain in the narrow hydrophobic channel delimited by the a and b subunits of NAAA9 In this structure, the polar head of PEA accommodates within a solvent exposed cleft comprising the catalytic residue, with the amide group making polar interactions with the oxyanion hole residues (Asn[292] and Glu[195] in hNAAA) and with the backbone of Asp[145]. A second set of simulations comprised MD runs of the complexes of rNAAA with PEA and with the other substrates, performed applying restraints of 0.5 kcal molÀ1 ÅÀ2 on the Ca atoms of the protein in order to maintain the overall architecture of the protein close to the X-ray structure, and carried out for 30 ns (compounds 1–3, 5–12, Figure 1) or 200 ns (4, 15, 16, Figure 1) in NVT conditions at 298 K. Specific rotation (1⁄2a2D0) was determined with a Perkin-Elmer 341 polarimeter

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Results

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