Amidase Signature Research Articles

A Novel Sulfatase for Acesulfame Degradation in Wastewater Treatment Plants as Evidenced from Shinella Strains.

The artificial sweetener acesulfame is a persistent pollutant in wastewater worldwide. So far, only a few bacterial isolates were recently found to degrade acesulfame efficiently. In Bosea and Chelatococcus strains, a Mn2+-dependent metallo-β-lactamase-type sulfatase and an amidase signature family enzyme catalyze acesulfame hydrolysis via acetoacetamide-N-sulfonate to acetoacetate. Here, we describe a new acesulfame sulfatase in Shinella strains isolated from wastewater treatment plants in Germany. Their genomes do not encode the Mn2+-dependent sulfatase. Instead, a formylglycine-dependent sulfatase gene was found, together with the acetoacetamide-N-sulfonate amidase gene on a plasmid shared by all known acesulfame-degrading Shinella strains. Heterologous expression, proteomics, and size exclusion chromatography corroborated the physiological function of the Shinella sulfatase in acesulfame hydrolysis. Since both acesulfame sulfatase types are absent in other bacterial genomes or metagenome-assembled genomes, we surveyed 73 tera base pairs of wastewater-associated metagenome raw data sets. Bosea/Chelatococcus sulfatase gene signatures were regularly found from 2013, particularly in North America, Europe, and East Asia, whereas Shinella sulfatase gene signatures were first detected in 2020. Moreover, signatures for the Shinella sulfatase and amidase genes co-occur only in six data sets from China, Finland, and Mexico, suggesting that the Shinella genes were enriched or introduced quite recently in wastewater treatment facilities.

High Salt-Resistant Urethanase Degrades Ethyl Carbamate in Soy Sauce.

Urethanase is a promising biocatalyst for degrading carcinogen ethyl carbamate (EC) in fermented foods. However, their vulnerability to high ethanol and/or salt and acidic conditions severely limits their applications. In this study, a novel urethanase from Alicyclobacillus pomorum (ApUH) was successfully discovered using a database search. ApUH shares 49.4% sequence identity with the reported amino acid sequences. It belongs to the Amidase Signature family and has a conserved "K-S-S" catalytic triad and the characteristic "GGSS" motif. The purified enzyme overexpressed in Escherichia coli exhibits a high EC affinity (Km, 0.306 mM) and broad pH tolerance (pH 4.0-9.0), with an optimum pH 7.0. Enzyme activity remained at 58% in 12% (w/v) NaCl, and 80% in 10% (v/v) ethanol or after 1 h treatment with the same ethanol solution at 37 °C. ApUH has no hydrolytic activity toward urea. Under 30 °C, the purified enzyme (200 U/L) degraded about 15.4 and 43.1% of the EC in soy sauce samples (pH 5.0, 6.0), respectively, in 5 h. Furthermore, the enzyme also showed high activity toward the class 2A carcinogen acrylamide in foods. These attractive properties indicate their potential applications in the food industry.

Biophysical Properties of Thermostable Amidase Produced by Aspergillus fumigatus in Submerged Fermentation

Amidases, also known as amidohydrolases (EC 3.5.1.4), are enzymes within the nitrilase superfamily or amidase signature family. They hydrolyze amides and nitriles into their corresponding acids while releasing ammonia. These enzymes are widely used in biotechnology and industry. However, their production is limited to a few organisms, which cannot meet industrial demand. Therefore, exploring new amidase sources is crucial. This study focuses on purifying and characterizing amidase from Aspergillus fumigatus using cold acetone precipitation and column chromatography with Sephadex G-100. The effects of temperature, pH and metal ions, on the enzyme activity were examined for both crude and purified amidase. The thermal and pH stability of the enzyme alongside the enzyme kinetics were also assessed. The purified amidase exhibited optimal activity of 13.1 U/mL at 70°C, with 65% residual activity after 5 hours at this temperature. The pH studies showed optimal activity at pH 5.0 for the purified enzyme (7.8 U/mL) and at pH 4.0 for the crude enzyme (5.5 U/mL). Metal ion effects indicated that Zn²⁺, Mg²⁺, and EDTA enhanced, while Cu²⁺ inhibited, the activities of both crude and purified amidase. Kinetic analysis showed that the enzyme followed Michaelis-Menten kinetics, with Km and Vmax values of 0.104 mM and 1.884 U/mL for crude amidase, and 0.017 mM and 1.872 U/mL for purified amidase. These findings suggest that amidase from Aspergillus fumigatus holds potential for industrial applications where amidases are essential.

Efficient Biodegradation of the Neonicotinoid Insecticide Flonicamid by Pseudaminobacter salicylatoxidans CGMCC 1.17248: Kinetics, Pathways, and Enzyme Properties.

Nitrile-containing insecticides can be converted into their amide derivatives by Pseudaminobacter salicylatoxidans. N-(4-trifluoromethylnicotinoyl) glycinamide (TFNG-AM) is converted to 4-(trifluoromethyl) nicotinoyl glycine (TFNG) using nitrile hydratase/amidase. However, the amidase that catalyzes this bioconversion has not yet been fully elucidated. In this study, it was discovered that flonicamid (FLO) is degraded by P. salicylatoxidans into the acid metabolite TFNG via the intermediate TFNG-AM. A half-life of 18.7 h was observed for P. salicylatoxidans resting cells, which transformed 82.8% of the available FLO in 48 h. The resulting amide metabolite, TFNG-AM, was almost all converted to TFNG within 19 d. A novel amidase-encoding gene was cloned and overexpressed in Escherichia coli. The enzyme, PmsiA, hydrolyzed TFNG-AM to TFNG. Despite being categorized as a member of the amidase signature enzyme superfamily, PsmiA only shares 20-30% identity with the 14 previously identified members of this family, indicating that PsmiA represents a novel class of enzyme. Homology structural modeling and molecular docking analyses suggested that key residues Glu247 and Met242 may significantly impact the catalytic activity of PsmiA. This study contributes to our understanding of the biodegradation process of nitrile-containing insecticides and the relationship between the structure and function of metabolic enzymes.

Recently evolved combination of unique sulfatase and amidase genes enables bacterial degradation of the wastewater micropollutant acesulfame worldwide.

Xenobiotics often challenge the principle of microbial infallibility. One example is acesulfame introduced in the 1980s as zero-calorie sweetener, which was recalcitrant in wastewater treatment plants until the early 2010s. Then, efficient removal has been reported with increasing frequency. By studying acesulfame metabolism in alphaproteobacterial degraders of the genera Bosea and Chelatococcus, we experimentally confirmed the previously postulated route of two subsequent hydrolysis steps via acetoacetamide-N-sulfonate (ANSA) to acetoacetate and sulfamate. Genome comparison of wildtype Bosea sp. 100-5 and an acesulfame degradation-defective mutant revealed the involvement of two plasmid-borne gene clusters. The acesulfame-hydrolyzing sulfatase is strictly manganese-dependent and belongs to the metallo beta-lactamase family. In all degraders analyzed, it is encoded on a highly conserved gene cluster embedded in a composite transposon. The ANSA amidase, on the other hand, is an amidase signature domain enzyme encoded in another gene cluster showing variable length among degrading strains. Transposition of the sulfatase gene cluster between chromosome and plasmid explains how the two catabolic gene clusters recently combined for the degradation of acesulfame. Searching available genomes and metagenomes for the two hydrolases and associated genes indicates that the acesulfame plasmid evolved and spread worldwide in short time. While the sulfatase is unprecedented and unique for acesulfame degraders, the amidase occurs in different genetic environments and likely evolved for the degradation of other substrates. Evolution of the acesulfame degradation pathway might have been supported by the presence of structurally related natural and anthropogenic compounds, such as aminoacyl sulfamate ribonucleotide or sulfonamide antibiotics.

Cloning and expression of the phenazine-1-carboxamide hydrolysis gene pzcH and the identification of the key amino acids necessary for its activity

Phenazine-1-carboxamide (PCN), a phenazine derivative, can cause toxicity risks to non target organisms. In this study, the Gram-positive bacteria Rhodococcus equi WH99 was found to have the ability to degrade PCN. PzcH, a novel amidase belonging to amidase signature (AS) family, responsible for hydrolyzing PCN to PCA was identified from strain WH99. PzcH shared no similarity with amidase PcnH which can also hydrolyze PCN and belong to the isochorismatase superfamily from Gram-negative bacteria Sphingomonas histidinilytica DS-9. PzcH also showed low similarity (˂ 39%) with other reported amidases. The optimal catalysis temperature and pH of PzcH was 30 °C and 9.0, respectively. The Km and kcat values of PzcH for PCN were 43.52 ± 4.82 μM and 17.028 ± 0.57 s−1, respectively. The molecular docking and point mutation experiment demonstrated that catalytic triad Lys80-Ser155-Ser179 are essential for PzcH to hydrolyze PCN. Strain WH99 can degrade PCN and PCA to reduce their toxicity against the sensitive organisms. This study enhances our understanding of the molecular mechanism of PCN degradation, presents the first report on the key amino acids in PzcH from the Gram-positive bacteria and provides an effective strain in the bioremediation PCN and PCA contaminated environments.

FAAH served a key membrane-anchoring and stabilizing role for NLRP3 protein independently of the endocannabinoid system.

NLRP3, the sensor protein of the NLRP3 inflammasome, plays central roles in innate immunity. Over-activation of NLRP3 inflammasome contributes to the pathogenesis of a variety of inflammatory diseases, while gain-of-function mutations of NLRP3 cause cryopyrin-associated periodic syndromes (CAPS). NLRP3 inhibitors, particularly those that inhibit inflammasome assembly and activation, are being intensively pursued, but alternative approaches for targeting NLRP3 would be highly desirable. During priming NLRP3 protein is synthesized on demand and becomes attached to the membranes of ER and mitochondria. Here, we show that fatty acid amide hydrolase (FAAH), the key integral membrane enzyme in the endocannabinoid system, unexpectedly served the critical membrane-anchoring and stabilizing role for NLRP3. The specific interaction between NLRP3 and FAAH, mediated by the NACHT and LRR domains of NLRP3 and the amidase signature sequence of FAAH, was essential for preventing CHIP- and NBR1-mediated selective autophagy of NLRP3. Heterozygous knockout of FAAH, resulting in ~50% reduction in both FAAH and NLRP3 expression, was sufficient to substantially inhibit the auto-inflammatory phenotypes of the NLRP3-R258W knock-in mice, while homozygous FAAH loss almost completely abrogates these phenotypes. Interestingly, select FAAH inhibitors, in particular URB597 and PF-04457845, disrupted NLRP3-FAAH interaction and induced autophagic NLRP3 degradation, leading to diminished inflammasome activation in mouse macrophage cells as well as in peripheral blood mononuclear cells isolated from CAPS patients. Our results unraveled a novel NLRP3-stabilizing mechanism and pinpointed NLRP3-FAAH interaction as a potential drug target for CAPS and other NLRP3-driven diseases.

Functional analysis, diversity, and distribution of carbendazim hydrolases MheI and CbmA, responsible for the initial step in carbendazim degradation.

Strains Rhodococcus qingshengii djl-6 and Rhodococcus jialingiae djl-6-2 both harbour the typical carbendazim degradation pathway with the hydrolysis of carbendazim to 2-aminobenzimidazole (2-AB) as the initial step. However, the enzymes involved in this process are still unknown. In this study, the previous reported carbendazim hydrolase MheI was found in strain djl-6, but not in strain djl-6-2, then another carbendazim hydrolase CbmA was obtained by a four-step purification strategy from strain djl-6-2. CbmA was classified as a member of the amidase signature superfamily with conserved catalytic site residues Ser157, Ser181, and Lys82, while MheI was classified as a member of the Abhydrolase superfamily with conserved catalytic site residues Ser77 and His224. The catalytic efficiency (kcat /Km ) of MheI (24.0-27.9μM-1 min-1 ) was 200 times more than that of CbmA (0.032-0.21 μM-1 min-1 ). The mheI gene (plasmid encoded) was highly conserved (>99% identity) in the strains from different bacterial genera and its plasmid encoded flanked by mobile genetic elements. The cmbA gene was highly conserved only in strains of the genus Rhodococcus and it was chromosomally encoded. Overall, the function, diversity, and distribution of carbendazim hydrolases MheI and CbmA will provide insights into the microbial degradation of carbendazim.

Features and application potential of microbial urethanases.

Urethanase (EC 3.5.1.75) can reduce ethyl carbamate (EC), a group 2A carcinogen found in foods and liquor. However, it is not yet commercially available. Urethanase has been detected as an intracellular enzyme from yeast, filamentous fungi, and bacteria. Based on the most recent progress in the sequence analysis of this enzyme, it was observed that amidase-type enzyme can degrade EC. All five enzymes had highly conserved sequences of amidase signature family, and their molecular masses were in the range of 52-62kDa. The enzymes of Candida parapsilosis and Aspergillus oryzae formed a homotetramer, and that of Rhodococcus equi strain TB-60 existed as a monomer. Most urethanases exhibited amidase activity, and those of C. parapsilosis and A. oryzae also demonstrated high activity against acrylamide, which is a group 2A carcinogen. It was recently reported that urease and esterase also exhibited urethanase activity. Although research on the enzymatic degradation of EC has been very limited, recently some sequences of EC-degrading enzyme have been elucidated, and it is anticipated that new enzymes would be developed and applied into practical use. KEY POINTS: • Recently, some urethanase sequences have been elucidated • The amino acid residues that formed the catalytic triad were conserved • Urethanase shows amidase activity and can also degrade acrylamide.

Beyond the Usual Suspects: Physiological Roles of the Arabidopsis Amidase Signature (AS) Superfamily Members in Plant Growth Processes and Stress Responses.

The diversification of land plants largely relies on their ability to cope with constant environmental fluctuations, which negatively impact their reproductive fitness and trigger adaptive responses to biotic and abiotic stresses. In this limiting landscape, cumulative research attention has centred on deepening the roles of major phytohormones, mostly auxins, together with brassinosteroids, jasmonates, and abscisic acid, despite the signaling networks orchestrating the crosstalk among them are so far only poorly understood. Accordingly, this review focuses on the Arabidopsis Amidase Signature (AS) superfamily members, with the aim of highlighting the hitherto relatively underappreciated functions of AMIDASE1 (AMI1) and FATTY ACID AMIDE HYDROLASE (FAAH), as comparable coordinators of the growth-defense trade-off, by balancing auxin and ABA homeostasis through the conversion of their likely bioactive substrates, indole-3-acetamide and N-acylethanolamine.

Biodegradation of the pyridinecarboxamide insecticide flonicamid by Microvirga flocculans and characterization of two novel amidases involved

Flonicamid (N-cyanomethyl-4-trifluoromethylnicotinamide, FLO) is a new type of pyridinecarboxamide insecticide that exhibits particularly good efficacy in pest control. However, the extensive use of FLO in agricultural production poses environmental risks. Hence, its environmental behavior and degradation mechanism have received increasing attention. Microvirga flocculans CGMCC 1.16731 rapidly degrades FLO to produce the intermediate N-(4-trifluoromethylnicotinoyl) glycinamide (TFNG-AM) and the end acid metabolite 4-(trifluoromethyl) nicotinol glycine (TFNG). This bioconversion is mediated by the nitrile hydratase/amidase system; however, the amidase that is responsible for the conversion of TFNG-AM to TFNG has not yet been reported. Here, gene cloning, overexpression in Escherichia coli and characterization of pure enzymes showed that two amidases—AmiA and AmiB—hydrolyzed TFNG-AM to TFNG. AmiA and AmiB showed only 20–30% identity to experimentally characterized amidase signature family members, and represent novel amidases. Compared with AmiA, AmiB was more sensitive to silver and copper ions but more resistant to organic solvents. Both enzymes demonstrated good pH tolerance and exhibited broad amide substrate specificity. Homology modeling suggested that residues Asp191 and Ser195 may strongly affect the catalytic activity of AmiA and AmiB, respectively. The present study furthers our understanding of the enzymatic mechanisms of biodegradation of nitrile-containing insecticides and may aid in the development of a bioremediation agent for FLO.

Study on the Activity of a New Amidase Signature (AS) Family

This study cloned a new amidase signature (AS) family gene from Escherichia coli (E. coii), and mainly investigated the activity of this enzyme. The protein, according to the bioinformatics analysis, belonged to the AS family. It was expressed, purified and characterized in E. coli BL21 (DE3). As a result, 40 °C and pH 8.0 were the optimum temperature and pH. It showed a wide substrate spectrum and high activity for aromatic and aliphatic amides, while with a narrow range of anilide substrate, and only propanol could be adopted as an effective substrate. Because of its wide range of substrate specificities, this will boost the applicability of amidase and make it have the potential for a wide range of applications in biosynthesis and biodegradation.

Structural insights into Pseudomonas aeruginosaType six secretion system exported effector 8

Recent reports indicate that the Type six secretion system exported effector 8 (Tse8) is a cytoactive effector secreted by the Type VI secretion system (T6SS) of the human pathogen Pseudomonas aeruginosa. The T6SS is a nanomachine that assembles inside of the bacteria and injects effectors/toxins into target cells, providing a fitness advantage over competing bacteria and facilitating host colonisation. Here we present the first crystal structure of Tse8 revealing that it conserves the architecture of the catalytic triad Lys84-transSer162-Ser186 that characterises members of the Amidase Signature superfamily. Furthermore, using binding affinity experiments, we show that the interaction of phenylmethylsulfonyl fluoride (PMSF) to Tse8 is dependent on the putative catalytic residue Ser186, providing support for its nucleophilic reactivity. This work thus demonstrates that Tse8 belongs to the Amidase Signature (AS) superfamily. Furthermore, it highlights Tse8 similarity to two family members: the Stenotrophomonas maltophilia Peptide Amidase and the Glutamyl-tRNAGln amidotransferase subunit A from Staphylococcus aureus.

An endocannabinoid catabolic enzyme FAAH and its paralogs in an early land plant reveal evolutionary and functional relationship with eukaryotic orthologs

Endocannabinoids were known to exist only among Animalia but recent report of their occurrence in early land plants prompted us to study its function and metabolism. In mammals, anandamide, as an endocannabinoid ligand, mediates several neurological and physiological processes, which are terminated by fatty acid amide hydrolase (FAAH). We identified nine orthologs of FAAH in the moss Physcomitrella patens (PpFAAH1 to PpFAAH9) with amidase signature and catalytic triad. The optimal amidase activity for PpFAAH1 was at 37 °C and pH 8.0, with higher specificity to anandamide. Further, the phylogeny and predicted structural analyses of the nine paralogs revealed that PpFAAH1 to PpFAAH4 were closely related to plant FAAH while PpFAAH6 to PpFAAH9 were to the rat FAAH, categorized based on the membrane binding cap, membrane access channel and substrate binding pocket. We also identified that a true ‘dynamic paddle’ that is responsible for tighter regulation of FAAH is recent in vertebrates and absent or not fully emerged in plants and non-vertebrates. These data reveal evolutionary and functional relationship among eukaryotic FAAH orthologs and features that contribute to versatility and tighter regulation of FAAH. Future studies will utilize FAAH mutants of moss to elucidate the role of anandamide in early land plants.

Fatty Acid Amide Hydrolases: An Expanded Capacity for Chemical Communication?

Fatty acid amide hydrolase (FAAH) is an enzyme that belongs to the amidase signature (AS) superfamily and is widely distributed in multicellular eukaryotes. FAAH hydrolyzes lipid signaling molecules - namely, N-acylethanolamines (NAEs) - which terminates their actions. Recently, the crystal structure of Arabidopsis thaliana FAAH was solved and key residues were identified for substrate-specific interactions. Here, focusing on residues surrounding the substrate-binding pocket, a comprehensive analysis of FAAH sequences from angiosperms reveals a distinctly different family of FAAH-like enzymes. We hypothesize that FAAH, in addition to its role in seedling development, also acts in an N-acyl amide communication axis to facilitate plant-microbe interactions and that structural diversity provides for the flexible use of a wide range of small lipophilic signaling molecules.

Comparison of Candida Albicans Fatty Acid Amide Hydrolase Structure with Homologous Amidase Signature Family Enzymes

Fatty acid amide hydrolase (FAAH) is a well-characterized member of the amidase signature (AS) family of serine hydrolases. The membrane-bound FAAH protein is responsible for the catabolism of neuromodulatory fatty acid amides, including anandamide and oleamide, that regulate a wide range of mammalian behaviors, including pain perception, inflammation, sleep, and cognitive/emotional state. To date, limited crystal structures of FAAH and non-mammalian AS family proteins have been determined and used for structure-based inhibitor design. In order to provide broader structural information, the crystal structure of FAAH from the pathogenic fungus Candida albicans was determined at a resolution of 2.2 Å. A structural comparison with a brown rat Rattus norvegicus FAAH as well as with other bacterial AS family members, MAE2 and PAM, showed overall similarities but there were several discriminative regions found: the transmembrane domain and the hydrophobic cap of the brown rat FAAH were completely absent in the fungal FAAH structure. Along with these results, a phylogenetic analysis of 19 species within the AS family showed that fungal FAAHs diverged from a common ancestor before the separation of eukarya and prokarya. Taken together, this study provides insights into developing more potent inhibitors of FAAH as well as expanding our knowledge of the relationships between AS family members.

Structure of the dihydrolipoamide succinyltransferase catalytic domain from Escherichia coli in a novel crystal form: a tale of a common protein crystallization contaminant.

The crystallization of amidase, the ultimate enzyme in the Trp-dependent auxin-biosynthesis pathway, from Arabidopsis thaliana was attempted using protein samples with at least 95% purity. Cube-shaped crystals that were assumed to be amidase crystals that belonged to space group I4 (unit-cell parameters a = b = 128.6, c = 249.7 Å) were obtained and diffracted to 3.0 Å resolution. Molecular replacement using structures from the PDB containing the amidase signature fold as search models was unsuccessful in yielding a convincing solution. Using the Sequence-Independent Molecular replacement Based on Available Databases (SIMBAD) program, it was discovered that the structure corresponded to dihydrolipoamide succinyltransferase from Escherichia coli (PDB entry 1c4t), which is considered to be a common crystallization contaminant protein. The structure was refined to an Rwork of 23.0% and an Rfree of 27.2% at 3.0 Å resolution. The structure was compared with others of the same protein deposited in the PDB. This is the first report of the structure of dihydrolipoamide succinyltransferase isolated without an expression tag and in this novel crystal form.

A Novel Amidase Signature Family Amidase from the Marine Actinomycete Salinispora arenicola CNS-205

We cloned a new gene from the amidase signature (AS) family, designated am , from the marine actinomycete Salinispora arenicola CNS-205. As indicated by bioinformatics analysis and site-directed mutagenesis, the AM (a gene encoding a putative amidase) protein belonged to the AS family. AM was expressed, purified, and characterised in Escherichia coli BL21 (DE3), and the AM molecular mass was determined to be 51 kDa. The optimal temperature and pH were 40 °C and pH 8.0, respectively. AM exhibited a wide substrate spectrum and showed amidase, aryl acylamidase, and acyl transferase activities. AM had high activity towards aromatic and aliphatic amides. The AM substrate specificity for anilides was very narrow, and only propanil could be used as an effective substrate. The extensive substrate range of AM indicates it may have broad potential applications in biosynthetic processes and biodegradation.

Characterization of the Propham Biodegradation Pathway in Starkeya sp. Strain YW6 and Cloning of a Novel Amidase Gene mmH

We previously isolated a monocrotophos-degrading strain Starkeya sp. YW6, which could also degrade propham. Here, we show that strain YW6 metabolizes propham via a pathway in which propham is initially hydrolyzed to aniline and then converted to catechol, which is then oxidized via an ortho-cleavage pathway. The novel amidase gene mmH was cloned from strain YW6 and expressed in Escherichia coli BL21(DE3). MmH, which exhibits aryl acylamidase activity, was purified for enzymatic analysis. Bioinformatic analysis confirmed that MmH belongs to the amidase signature (AS) enzyme family and shares 26-50% identity with several AS family members. MmH (molecular mass of 53 kDa) was most active at 40 °C and pH 8.0 and showed high activity toward propham, with Kcat and Km values of 33.4 s-1 and 16.9 μM, respectively. These characteristics make MmH suitable for novel amide biosynthesis and environmental remediation.

FAAH-Catalyzed C-C Bond Cleavage of a New Multitarget Analgesic Drug.

The discovery of extended catalytic versatilities is of great importance in both the chemistry and biotechnology fields. Fatty acid amide hydrolase (FAAH) belongs to the amidase signature superfamily and is a major endocannabinoid inactivating enzyme using an atypical catalytic mechanism involving hydrolysis of amide and occasionally ester bonds. FAAH inhibitors are efficacious in experimental models of neuropathic pain, inflammation, and anxiety, among others. We report a new multitarget drug, AGN220653, containing a carboxyamide-4-oxazole moiety and endowed with efficacious analgesic and anti-inflammatory activities, which are partly due to its capability of achieving inhibition of FAAH, and subsequently increasing the tissue concentrations of the endocannabinoid anandamide. This inhibitor behaves as a noncompetitive, slowly reversible inhibitor. Autoradiography of purified FAAH incubated with AGN220653, opportunely radiolabeled, indicated covalent binding followed by fragmentation of the molecule. Molecular docking suggested a possible nucleophilic attack by FAAH-Ser241 on the carbonyl group of the carboxyamide-4-oxazole moiety, resulting in the cleavage of the C-C bond between the oxazole and the carboxyamide moieties, instead of either of the two available amide bonds. MRM-MS analyses only detected the Ser241-assisted formation of the carbamate intermediate, thus confirming the cleavage of the aforementioned C-C bond. Quantum mechanics calculations were fully consistent with this mechanism. The study exemplifies how FAAH structural features and mechanism of action may override the binding and reactivity propensities of substrates. This unpredicted mechanism could pave the way to the future development of a completely new class of amidase inhibitors, of potential use against pain, inflammation, and mood disorders.