Skip to main content

Characterization, comparative, and functional analysis of arylacetamide deacetylase from Gnathostomata organisms



Arylacetamide deacetylase (AADAC) is a lipolytic enzyme involved in xenobiotic metabolism. The characterization in terms of activity and substrate preference has been limited to a few mammalian species. The potential role and catalytic activities of AADAC from other organisms are still poorly understood. Therefore, in this work, the physicochemical properties, proteomic analysis, and protein-protein interactions from Gnathostomata organisms were investigated.


The analysis were performed with 142 orthologue sequences with ~ 48–100% identity with human AADAC. The catalytic motif HGG[A/G] tetrapeptide block was conserved through all AADAC orthologues. Four variations were found in the consensus pentapeptide GXSXG sequence (GDSAG, GESAG, GDSSG, and GSSSG), and a novel motif YXLXP was found. The prediction of N-glycosylation sites projected 4, 1, 6, and 4 different patterns for amphibians, birds, mammals, and reptiles, respectively. The transmembrane regions of AADAC orthologues were not conserved among groups, and variations in the number and orientation of the active site and C-terminal carboxyl were observed among the sequences studied. The protein-protein interaction of AADAC orthologues were related to cancer, lipid, and xenobiotic metabolism genes.


The findings from this computational analysis offer new insight into one of the main enzymes involved in xenobiotic metabolism from mammals, reptiles, amphibians, and birds and its potential use in medical and veterinarian biotechnological approaches.

Graphical Abstract


Arylacetamide deacetylase (AADAC) is a lipolytic enzyme involved mainly in the hydrolysis of esters and amides [1,2,3]. Its active site domain is a classic lipase/esterase GXSXG motif (catalytic triad Ser189, His373, and Asp343), sharing high sequence homology with the active site of hormone-sensitive lipases (HSL). Although AADAC was initially considered as an esterase, current findings suggest that AADAC could be classified as a lipase, due to the strong homology shared with HSL, the acceptance of water-insoluble substrates, and the observed inhibition by classic lipase inhibitors, such as E600 (diethyl-p-nitrophenyl phosphate) [4, 5].

AADAC strongly contributes to drug hydrolysis, resulting in xenobiotic detoxification or prodrug activation [1]. The catalytic activity of AADAC has been tested on the hydrolysis and deacetylation on a wide variety of prodrugs, such as flutamide, phenacetin, indiplon, ketoconazole, rifabutin, rifampicin, and rifapentine [2, 6,7,8,9]. In contrast to other transmembrane enzymes, such as carboxylesterase 1 (CES1), carboxylesterase 2 (CES2), and paraoxonase 1 (PON1), AADAC selectively prefers bulky and small acyl moieties, such as fluorescein diacetate, N-monoacetyldapsone, and propanil [1]. Similarly, AADAC has been linked to the hydrolysis and activation of abiraterone acetate, a prodrug for metastatic castration-resistant prostate cancer [10].

Although in humans, AADAC is primarily expressed in the lumen side of the endoplasmic reticulum (ER) of the liver and gastrointestinal tract (jejunum, small intestine, and colon) [11, 12], its expression has also been found in the brain, particularly in the Purkinje cell layer of the human cerebellum, hippocampus, corpus callosum, and caudate nucleus [13]; adrenal cortex/medulla, and pancreas [14]. Due to its vast location, the physiological effects linked to an upper or lower expression or inhibition of AADAC could be beneficial, e.g., the upregulation of AADAC expression contributed to a better prognosis for several types of cancer [15,16,17,18,19]. On the other hand, variants or deletions of the AADAC gene have been linked to increased susceptibility to suffer Tourette syndrome [13].

In drug development, the extrapolation of data from orthologues is used to estimate the pharmacological effects and toxicity of drug candidates [20]. Orthologues are genes that evolved from a common ancestor by speciation; therefore, orthologue proteins are likely to have similar biological roles [21]. This similarity allows the identification of enzymes from organisms other than model organisms, such as birds, reptiles, or amphibians. Some AADAC orthologues, such as mouse, dog, rat, and cynomolgus macaque AADAC have been studied for their substrate recognition and specificity [11, 12, 22,23,24]. For example, human AADAC can hydrolyze flutamide, phenacetin, indiplon, and rifampicin, whereas rat and mouse AADAC only hydrolyzes flutamide and phenacetin, and not rifampicin [11]. In contrast, dog AADAC only hydrolyzes phenacetin and not indiplon [12]. Differences in enzyme location are also observed among orthologue AADAC. For instance, in human and cynomolgus macaques, AADAC mRNA is found in the liver and small intestine, whereas in rats and mice, mRNA AADAC is expressed in the kidney [22].

Mice and rats are the preferred organisms for toxicological studies, and differences in substrate preference by human, rat, and mouse liver microsomes expressing AADAC have also been observed [2]. Human and rat liver microsomes could hydrolyze indiplon, but mouse liver microsomes could not, whereas human and mouse liver microsomes hydrolyzed phenacetin and ketoconazole. Interestingly, neither mouse nor rat liver microsomes had any activity on rifamycins, whereas human liver microsomes showed activity values from 20 to 60 pmol/min/mg.

Given the importance of human AADAC in lipid, endobiotic, and xenobiotic metabolism, the present work focused on the functional analysis of AADAC orthologues, human AADAC-protein interactions, and physicochemical properties of a series of AADAC orthologue sequences from mammals, reptiles, amphibians, and birds.


Collection of AADAC protein sequences

AADAC amino acid sequences were retrieved from GenBank and Ensembl databases through the Basic Local Alignment Search Tool (BLAST). Searches were performed using the annotated Human Arylacetamide Deacetylase amino acid sequence (GenBank accession no. NP_001077.2, and Ensembl accession no. ENSG00000114771), and InterProScan was used to confirm the protein domains [25]. The orthologue sequences were selected according to the following parameters: (i) sequences with lengths between 393 and 415 amino acids; (ii) sequences without gaps, unspecific amino acids, or truncated regions, (iii) sequences containing the α/β hydrolase-3 domain, and (iv) sequences belonging to mammals, amphibians, birds, and reptiles.

Phylogenetic analysis

The phylogenetic tree was constructed using an amino acid alignment of human AADAC orthologues with MUSCLE in Mega X Software (RRID:SCR_000667) [26] with the following settings: (i) hydrophobicity multiplier (1.20), (ii) max iterations (16), and (iii) cluster method (UPGMA), also, the distances were calculated using the Jones-Taylor-Thornton matrix (JTT), and the tree was created with the maximum likelihood method with 1000 bootstrap replicates. Finally, the Interactive Tree of Life (iTOL v6; was employed to display the tree [27]. The Fitch (or similarity) matrix of the 142 orthologues was developed in RStudio software (v2022.07.2) using the R package SeqinR 1.0-2: Biological Sequences Retrieval and Analysis [28]. A protein alignment was used to calculate the pairwise distance matrix of aligned sequences using the function dist.alignment (x, matrix = similarity). The Numerical Taxonomy Multivariate Analysis System (NTSYSpc v2.02) software calculated the correlation matrix of amino acid sequences coding for the AADAC gene using the Dis/similarity module for qualitative data. Principal component analysis (PCA) was performed by plotting the 2-dimensional (2D) Dis/similarity matrix in NTSYSpc.

AADAC-protein interaction prediction

Potential protein-protein association networks for human AADAC were predicted with the STRING online database (RRID:SCR_005223) [29], whereas the protein-protein association for AADAC orthologues was predicted with the BioGRID database ( (RRID:SCR_007393) [30]. For the STRING database (, the sequences of AADAC were selected according to the above criteria. For the interaction analysis in BioGRID, the target-interacting protein data from human AADAC, paraoxonase 1 (PON1), carboxylesterase 1 (CES1), carboxylesterase 2 (CES2), carboxylesterase 3 (CES3) were used.

Motif search

The lipase/esterase consensus motifs GXSXG and HGGG were inferred by sequence alignment with human AADAC and hormone-sensitive lipase (HSL, GenBank accession no. AAA69810.1). The logo plot representations were generated using Logomaker [31], and WebLogo [32]. The code that creates the Logomaker plots is available on GitHub at:

To analyze the conserved motifs in AADAC orthologue proteins, 10 putative motifs between 6 and 50 amino acids were predicted with the MEME Suite 5.4.1 [33].

Modeling of three-dimensional AADAC structures and predicted physicochemical properties and transmembrane regions

Protein modeling of AADAC was performed with AlphaFold via the Google Colab platform [34], and visualized with UCSF Chimera software [35]. The physicochemical parameters of human AADAC orthologues were predicted with ProtParam using the EXPASY server (RRID:SCR_018087) [36]. The sequences in FASTA were used to analyze the molecular weight (MW), theoretical pI, the total number of negatively and positively charged residues, amino acid composition, extinction coefficients, instability index, aliphatic index, and grand average of hydropathicity (GRAVY). In addition, the transmembrane regions were inferred with TMHMM 2.0 (RRID:SCR_014935) [37], and glycosylations were predicted with NetOGlyc 4.0 (RRID:SCR_009026) [38]. One-way non-parametric ANOVA was used to evaluate the significative differences between orthologues characteristics using GraphPad Prism version 8.0.2 (RRID:SCR_002798).


A total of 142 gene sequences from mammals (67), amphibians (4), birds (53), and reptiles (18) were collected from GenBank and confirmed with Ensembl database (Table 1) as orthologues. AADAC protein sequences showed ~ 48–100% identity across orthologues when human AADAC (NP_001077.2) was used as reference. In all cases, the α/β hydrolase domain was located between residues 107-376. Similarly, the amino acid residues of the catalytic triad of lipases/esterases (Ser, Asp/Glu, and His) were conserved among all the orthologues studied (Supplementary Figure S1).

Table 1 Structural characteristics from AADAC orthologues

The phylogenetic relationships of AADAC orthologues are shown in Fig. 1. The avian taxon had a common ancestor from which descended two distinctive subgroups: (i) clades XII and XIII, and (ii) clades VIII to XI. Under these terms, it is inferred that birds have the lowest percentage of identity with human AADAC. The analysis shows a marked difference between AADAC from the selected organisms. In mammals, four distinctive clades were observed: in clade I; human AADAC shared a common ancestor with AADAC from Pan paniscus and Pan troglodytes; in clade II, mouse and rat AADAC were grouped; clade III belonged to dog AADAC, whereas camels and hedgehogs were grouped in clade IV. Amphibians, represented by clade V, and clade IV from mammals evolved from a common ancestor. Reptiles, divided into clades VI and VII, share a common ancestor. PCA shows the similarity between orthologs of different taxa (Fig. 2A, B). It can be observed that organisms of the same taxon share more significant similarities (Fig. 2B), comparable to that observed in the phylogenetic tree (Fig. 1).

Fig. 1
figure 1

Maximum Likelihood tree showing the phylogenetic relationships of AADAC orthologues. Mammal AADAC sequences are distributed in clades. Sequences from mammals are highlighted in blue, amphibians in lilac, reptiles in green, and birds in pink. Distances were calculated using the Jones-Taylor-Thornton matrix (JTT), and the tree was created with the Maximum Likelihood method with 1,000 bootstrap replicates

Fig. 2
figure 2

Principal component analysis. A The Fitch matrix of the 142 orthologues. B Correlation matrix of the amino acid sequences coding for the AADAC genes, the Dis/similarity matrix was created in NTSYSpc; amphibian organisms are represented in purple, avian organisms are represented in pink, mammalian organisms are represented in blue, and reptiles in green

Human AADAC is closely related to three hydrolytic enzymes belonging to the carboxylesterase family: CES1, CES2, and CES3, which are ER-linked enzymes that contain the α/β hydrolase domain and are primarily known to be involved in phase I of drug or xenobiotic metabolism [39] . A single interactome to relate the genetic and physical interactions of CES1, CES2, PON1, and human AADAC proteins/genes was constructed (Fig. 3). The amyloid-beta precursor protein (APP) shows a chemical-protein interaction with CES2, AADAC, CES1, and PON1 (Fig. 3).

Fig. 3
figure 3

BioGRID-base human AADAC-protein interaction network

In humans, APP is a cell surface protein with signal transduction properties that control the viability, proliferation, migration, and aggressiveness of various cancer cells, such as breast cancer, ovarian cell adenocarcinoma, medulloblastoma, and neuroblastoma [32]. Additionally, APP interacted with Amyloid-Beta Precursor Protein Binding Protein 2 (APPBP2), which is involved in non-small cell lung cancer (NSCLC), a type of cancer accountable for ~ 80% of all lung carcinoma patients [33]. On the other hand, the interactions of 70 AADAC orthologues were analyzed using the STRING database (Supplementary Table S2). From the sequences studied, 45 AADAC orthologues were related to dihydrodiol dehydrogenases (DHDH), 57 sequences showed an interaction with the succinate receptor 1 (SUCNR1), and 44 interacted with ATP8B3. DHDH is involved in the detoxication of trans-dihydrodiol and carcinogenic metabolites of polycyclic aromatic hydrocarbons [40]; SUCNR1 is related to conditions such as hypertension, diabetes, and obesity [41], whereas ATP8B3 is involved in aminophospholipid transport in the liver [42].

The structural motif of the consensus pentapeptide GXSXG (Table 1 and Fig. 4A) was illustrated using consensus sequence logos grouped by amphibians, birds, mammals, and reptiles. Four different residues were found occupying the X position of the GXSXG sequence: GDSAG (92%), GESAG (2.1%), GDSSG (2.8%), and GSSSG (1.4%), while the HGG[G/A] tetrapeptide block was conserved through all AADAC orthologues, being the G/Gly residue following the HGG motif (HGGG) extensively conserved (99.2%) (Fig. 4B and Fig. 5C/motif 1). The search for structural motifs using the MEME-suite (motif-based sequence analysis tools) displayed 10 essential motifs for all AADAC sequences studied. Motifs 1, 3, 6, 8, and 9 were absent in star-nosed mole (Condylura cristata), arctic ground squirrel (Urocitellus parryii), yellow-bellied marmot (Marmota falviventris), chicken (Gallus gallus), gecko (Gekko japonicus), maiden ray (Amblyraja radiata), small tree finch (Camrahynchus parvulus), gaboon caecilian (Geotrypetes seraphini), and little egret (Egretta garzetta) (Fig. 5 and Supplementary Figure S2). Motif 7 (Fig. 5) corresponds to the transmembrane region, while motifs 1, 3, had glycosylation sites such as NVTV, NWSS, and NWSN. A possibly important region identified in motif 5 is the YXLXP sequence, where X represents the amino acids R/Arg or A/Ala present in all orthologues (Supplementary Figure S1, Fig. 5/motif 5), and forms a β-α loop in all orthologues sequences studied (Fig. 5A, B). The AADAC folding among organisms with 10 motifs was similar to human (Fig. 5C), and sequences with any missing motif showed structural differences, mainly in the formation of alpha helices and beta strands (Fig. 6D, F).

Fig. 4
figure 4

Conserved AADAC motifs using LogoMaker (A) and WebLogo (B and C). In all cases, the amino acid number indicated in the image is relative to the human AADAC sequence. A Logo showing the conserved GXSXG motif, which depicts the catalytic Ser, located in residues 113 to 126. B Logo showing the conserved HGGG motif, located in residues 113 to 126. C Logo showing the conserved transmembrane region of AADAC orthologues, located in residues 1 to 25

Fig. 5
figure 5

Conserved motifs in AADAC orthologues using MEME-suite. A Ten representative conserved motifs. Motif 1 (red), motif 2 (cyan), motif 3 (green), motif 4 (violet), motif 5 (orange), motif 6 (lime), motif 7 (blue), motif 8 (fuchsia), motif 9 (dark orange), and motif 10 (yellow). B The locations along the sequence of the 10 representative motifs depicted in A. The complete list is illustrated in Supplementary Figure S2. C Representation of all motifs of AADACs orthologues. The HGGG and GXSXG motifs are highlighted in gray. N and C, at the bottom of the images represent the amino terminal and carboxyl terminal

Fig. 6
figure 6

Tridimensional structural comparison of AADAC from orthologues. A ADDAC from Homo sapiens, the active site is shown in red, and the YXLXP motif in orange. B Active site of AADAC from Homo sapiens. The catalytic amino acids are represented by sticks. C Structural alignment of representative organisms from mammals (Macaca mulata in aqua), amphibian (Nanorana parkeri in pink), birds (Anas platyrhynchos in purple), reptiles (Trachemys scripta elegans in light green). D Structural alignment of mammalian AADAC showcasing the motif differences (Condylura cristata in grey color, Urocitellus parryii in salmon color and Marmota flaviventris in yellow color). E Structural alignment of birds (Camarhynchus parvulus in gold color, Gallus gallus in steel blue and Egretta garzetta in brown). F Structural alignment of amphibian and reptile organisms with motif differences (Amblyraja radiata in pink, Geotrypetes seraphini in aquamarine and Gekko japonicus in beige). The numbers indicate the structural motif related to MEME suite analysis (Fig. 3)

The physicochemical characterization shows that the molecular mass among orthologs remains between 44 and 46 kDa; however, for mammals, the mean is 45.8 kDa, while for birds and amphibians, it is 45.0 and 45.8 kDa, respectively. The number of negatively charged residues (Asp + Glu) was greater than the number of positively charged residues (Arg + Lys). The sum of hydropathy values of all amino acids divided by protein length showed similar tendencies between orthologues. The instability index is between 31 and 62 h, and some birds orthologues showed the most stable AADAC (Fig. 7). The prediction of N-glycosylation sites with NetNGlyc-4.0 server projected 4, 1, 6, and 4 different patterns for amphibians, bird, mammals, and reptiles, respectively.

Fig. 7
figure 7

Distribution plot depicting seven major predicted physicochemical properties of AADAC orthologues using the ProtParam Tool. A Molecular mass (kDa), B Percentage of negatively charged amino acids (Asp + Glu), C Percentage of positively charged amino acids (Arg + Lys), D Theoretical isoelectric point (pI), E Grand average of hydropathicity index (GRAVY), F Instability index, and G Aliphatic index. The complete computed physicochemical properties for each AADAC ortholog are depicted in Supplementary Table S1. One-way non-parametric ANOVA was used to evaluate significant differences between orthologues characteristics using GraphPad Prism version 8.0.2

The transmembrane regions of AADAC orthologues varied between 0 and 3. From 142 orthologue sequences, 48 organisms did not show transmembrane regions (33.8%), 73 organisms showed a single transmembrane region with the C-terminal oriented towards the lumen (51.4%), 5 organisms had 2 transmembrane regions with the active site towards the cytosol (3.5%), and 16 organisms showed 3 transmembrane regions with the C-terminal carboxyl oriented towards the ER lumen (11.2%) (Table 1).


The phylogenetic history of human AADAC orthologues infers those common ancestral sequences evolved in the avian taxon, with speciation events occurring in a descending fashion in reptile, amphibian, and mammalian taxa, respectively.

Only nine AADAC orthologues showed interactions with carboxyl esterases (CES), belonging to red fox (Vulpes vulpes), Bolivian squirrel monkey (Samiri boliviensis), brown rat (Rattus norvegicus), house mouse (Mus musculus), Mus pahari, shrew-like mouse (Mus coroli), Mongolian gerbil (Meriones unguiculatus), one-humped camel (Camelus dromedarius), and human (Homo sapiens). It can be inferred that these nine AADAC may also be able to hydrolyze human AADAC substrates, such as xenobiotics.

The consensus sequence GDSAG found in mammals, birds, reptiles, and amphibians belongs to family IV of α/β hydrolases, also known as HSL family. The GESAG motif, found only in three orthologues, gray seal (Halichoerus grypus), common seal (Phoca vitulina), and weddell seal (Leptonychotes weddellii)), belonged to family VII of α/β hydrolases [43]. Three AADAC orthologues belonging to the order of Crocodilia (Alligator sinensis, Crocodylus porosus, and Gavialis gangeticus) and shrewmouse (Mus pahari) showed the GDSSG consensus sequence. The natal long-fingered bat (Miniopterus natalensis) and the black flying fox (Pteropus alecto) were the only AADAC orthologues showing the consensus pentapeptide GSSSG (Table 1 and Supplementary Figure S1).

The HGGG motif forms a loop close to the active site and directly participates in the catalytic hydrolysis by stabilizing the oxyanion intermediate of the reaction. The presence of multiple G/Gly residues confers greater flexibility to this loop. In contrast, some studies point that a change in the last G/Gly residue for an A/Ala decreases the enzyme activity up to 40% [44]. Pteropus alecto AADAC was the only orthologue to show an A/Ala residue placed at the last position (HGGA). The YXLXP motif has been previously reported in the telomeric protein SNM1B/Apollo (a member of the Metallo-β-lactamase/β-CASP family), which is involved in binding to TRF2 (telomeric repeat-binding factor 2) [45]; Similarly, SLX4 protein involved in DNA repair, contains a H/YXLXP motif and binds human TRF2. A similar site in the TRFH domain of TRF1 (around residue F142), FXLXP motif, serves as an anchor to TIN2 protein (TRF1-interacting protein 2) [46, 47].

Another essential structural region is the transmembrane region (Fig. 3C). The amino acids belonging to this region are responsible for anchoring the protein to the lipid bilayer. The most conserved amino acids among the studied orthologues were Y/Tyr, P/Pro, G/Gly, and K/Lys. In contrast, amino acids at positions 3, 5, 11, 12, 15, 21, and 23 showed increased variability among AADAC orthologues.

The predicted physicochemical characteristics of AADAC orthologues are resumed in the Supplementary Table S1. The calculated Molecular weight (MW) of AADAC orthologues is shown for amphibians, birds, mammals, and reptiles, and the minimum and maximum values are highlighted using a box and whisker plot (Fig. 7A). Human AADAC has been previously purified and characterized from liver tissue. Its MW was deduced at 45.671 kDa by gel electrophoresis [4], which is consistent with the MW value obtained by ProtParam for human AADAC (45.73 kDa), and similar to the average MW value calculated for mammals (45.68 kDa). The highest MW value belonged to AADAC from reptiles (46.77 kDa), while AADAC from birds had an average of 45.1 kDa, being the taxa with the lowest MW values.

The amount of Asp + Glu and Arg + Lys is related to the isoelectric point of the protein (Fig. 5B, C). Human AADAC orthologues showed isoelectric point (pI) values between 5.5 and 9.3 (Fig. 5D and Supplementary Table S1). The theoretical pI of human AADAC was 8.75, which differed from the pI calculated by in vivo experiments (9.36) [4]. Mammals showed pI values ranging from 6.0 to 9.3, followed by birds (5.6–9.2), reptiles (5.5–8.5), and amphibians (6.0–8.3).

The GRAVY value for a protein or a peptide is the sum of hydropathy values of all amino acids divided by the protein length [48]. Positive GRAVY values indicate hydrophobicity, whereas negative values mean hydrophilicity. The GRAVY value for human AADAC was found to be negative, implying a hydrophilic character, and similar GRAVY results were observed for mammals, amphibians, and reptiles AADAC (Fig. 7E). In contrast, birds were the only taxa showing positive GRAVY values, thus indicating hydrophobicity.

The instability index of a protein is calculated based on the presence of certain dipeptides. If the instability index value of a given protein is less than 40, it is considered stable under atmospheric conditions, and have a longer in vivo life span (~ 16 h), whereas proteins with an instability index > 40 show an in vivo half-life of less than 5 h [49]. The instability index value of human AADAC was 33.96 (Fig. 7E), which is considered a stable protein for in vivo experiments. In fact, human AADAC has already been expressed in sf9, DH10Bac, and E. coli cells, further purified and characterized for drug hydrolysis assays [6, 50, 51]. The average instability index values of AADAC orthologues ranged from 40 to 45 (Fig. 7F). Different model organisms for toxicological studies found in the mammalian taxon, such as mice, rats, and dogs, showed a protein instability index > 40, thus classifying them as in vivo unstable with a life span of fewer than five hours. Despite these values, mouse, rat, and dog AADAC microsomes have been successfully used in vitro to hydrolyze various prodrugs, confirming their role in the metabolism of the already known substrates of human AADAC [11, 12, 52].

For human AADAC, the high aliphatic index value was 99.15, which is considered thermostable. In general, all the AADAC orthologues studied showed aliphatic index values above 86.22, indicating a reliable degree of thermostability (Fig. 7G).

The potential N-glycosylation sites of amphibian, bird, mammal, and reptile AADAC were predicted with the NetNGlyc-4.0 server, and the results are detailed in Table 1. For N-glycosylation, an N-acetylglucosamine residue is linked via an amide bond to an asparagine (N/Asn) from the consensus sequence NX(S/T), where X can be any amino acid except proline (P/Pro). The presence of the consensus sequence is required for N-linked glycosylation; however, the potential sites might not be glycosylated [53]. For amphibians, the neuronal prediction gave two main glycosylation sites. All four amphibian sequences showed glycosylation sites at 78(NVTV/I). For bird AADAC, 39 sequences had one glycosylation site (78(NVTV), 73.6% of sequences), 12 sequences had two glycosylation sites (78(NVTV)-282(NWS[Q/R/V/L/N]) or 78(NVTV)-88(N[V/T/I][S/T]V), 22.6% of sequences), whereas the sequence of the bird Neopelma chrysocephalum showed no glycosylation site.

Mammalian AADAC showed six different glycosylation patterns. The most common pattern was 78(NV/ITV)-282(NWSS/A) (36 sequences in total, 53.7%), including AADAC sequences of Homo sapiens, Pan paniscus, Pan troglodytes, and Canis lupus familiaris. This is in agreement with the results reported in the literature [52], where their findings proved that N-glycosylation at the N282 residue of human AADAC was crucial for enzymatic activity by proper folding. The following observed patterns were 77(NVTV) (7 sequences, 10.4%, including Rattus rattus and Rattus norvegicus), and 282(NWSS) (5 sequences, 7.5%), and 77(NVTV)-281(NWSS) (4 sequences, 6%). Three glycosylation sites were found for four mammal sequences (78(NITV)-281(NWSS)-298(NRTY), 78(NVTV)-282(NWSS)-302(NGTS), 56(NHSM)-78(NVTV)-282(NWSS), and 55(NHSM)-77(NVTV)-281(NWSS), whereas 11 mammal sequences showed other glycosylation patterns. The AADAC sequence of Mus musculus had no N-glycosylation sites.

The prediction of N-glycosylation sites for reptile AADAC projected four different patterns. Six reptile sequences (33.3%) had the 282(NWS/N/R/H/K/E) site, 4 sequences (22.2%) had the 78(NVTV)-282(NWSN) site, 5 sequences (27.8%) showed different N-glycosylation patterns, and 3 sequences (16.7%) had no N-glycosylations.

In humans, AADAC is a type II transmembrane protein comprising a short, positively charged N-terminal region oriented towards the cytoplasm, a single transmembrane region, and a C-terminal tail oriented towards the lumen of the ER, containing the catalytic triad Ser189, Asp343, and His373 [5, 52, 54]. The lumen location of the catalytic triad was confirmed by the hydrolysis of D-13223, a substrate that is not observed in human liver cytosol [55].

The transmembrane regions of human AADAC and its orthologues were predicted with TMHMM (Table 1). TMHMM has a reliability of soluble and transmembrane proteins of 70–80% of assertiveness [37]. The server predicted a transmembrane helix located at residues 5–24 (Table 1), and an outer sequence of amino acid 25-399, which is consistent with the literature [54]. The active site of human AADAC faces the lumen site of the endoplasmic reticulum (RE), which implies different catalytic capacities compared to other carboxylesterases [5]. For example, substrates for cytosolic lipases, such as glycerolphospholipids or triglycerides stored in cytoplasmic lipid droplets, cannot cross the ER membrane and therefore are not hydrolyzed by AADAC [5].

All studied orthologues shared the same amino-terminal location at the cytosol, whereas the C-terminal was oriented towards the lumen of the RE.


Although AADAC is a ubiquitous protein spread among organisms from different taxa, drug hydrolysis assays and toxicological studies are limited to a few species. The predicted physicochemical characteristics of the studied 142 orthologues show similarity among the groups; however, the composition of amino acids as Arg + Lys and Asp + Glu ratios differed among the studied organisms, which generates differences between the isoelectric point. The orthologues were related to DHDH, ATP8B3, and P2RY1 genes, expressed in the lipid membrane, and directly related to skin and lung cancer genes (APP and APPBP2), suggesting that they may share mechanisms among these diseases. In addition, the comparative analysis of AADAC orthologues revealed that the sequence YXLXP, which has been involved as DNA repair mechanism, was shared by all the organisms studied. Other structural motifs that are fingerprints of lipolytic enzymes (GXSXG and HGGG) were found in all orthologs; however, according to the predicted 3D structures, missing motifs can generate conformational changes that can affect enzyme-substrate interaction.

The number of transmembrane regions varied among organisms and did not follow a pattern among mammals, amphibians, birds, or reptiles. Some substrate selectivities among the studied orthologues could be inferred by the phylogenetic tree constructed; however, further biochemical studies are mandatory to corroborate the results herein presented. This information, together with the analysis of the interactome, offers an overview of how these AADAC from the studied organisms could interact with various drugs tested mainly in primates or rodents. The collected data shown in this work broadens the knowledge of AADAC orthologues and could help to better select new model organisms as future candidates for detoxification, xenobiotic processing, and drug disposition studies.

Availability of data and materials

The manuscript has data included as electronic supplementary material.



Arylacetamide deacetylase


Hormone-sensitive lipases


Diethyl-p-nitrophenyl phosphate


Carboxylesterase 1


Carboxylesterase 2


Paraoxonase 1


Basic Local Alignment Search Tool


Molecular weight


  1. Fukami T, Kariya M, Kurokawa T, Iida A, Nakajima M (2015) Comparison of substrate specificity among human arylacetamide deacetylase and carboxylesterases. Eur J Pharm Sci 78:47–53.

    Article  Google Scholar 

  2. Kisui F, Fukami T, Nakano M, Nakajima M. Strain and sex differences in drug hydrolase activities in rodent livers. Eur J Pharm Sci 2020;142:105143.

  3. Morikawa T, Fukami T, Gotoh-Saito S, Nakano M, Nakajima M. PPARα regulates the expression of human arylacetamide deacetylase involved in drug hydrolysis and lipid metabolism. Biochem Pharmacol 2022;199:115010.

  4. Probst MR, Beer M, Beer D, Jenö P, Meyer UA, Gasser R (1994) Human liver arylacetamide deacetylase. Molecular cloning of a novel esterase involved in the metabolic activation of arylamine carcinogens with high sequence similarity to hormone-sensitive lipase. J Biol Chem 269:21650–21656

    Article  Google Scholar 

  5. Lo V, Erickson B, Thomason-Hughes M, Ko KWS, Dolinsky VW, Nelson R, et al. Arylacetamide deacetylase attenuates fatty-acid-induced triacylglycerol accumulation in rat hepatoma cells. J Lipid Res 2010;51:368–377.

  6. Hirosawa K, Fukami T, Tashiro K, Sakai Y, Kisui F, Nakano M, et al. Role of human AADAC on hydrolysis of eslicarbazepine acetate and effects of AADAC genetic polymorphisms on hydrolase activity. Drug Metab Dispos 2021;49.

  7. Probst MR, Jenö P, Meyer UA. Purification and characterization of a human liver arylacetamide deacetylase. Biochem Biophys Res Commun 1991;177:453–459.

  8. Nagaoka M, Fukami T, Kisui F, Yamada T, Sakai Y, Tashiro K, et al. Arylacetamide deacetylase knockout mice are sensitive to ketoconazole-induced hepatotoxicity and adrenal insufficiency. Biochem Pharmacol 2022;195:114842.

  9. Phaisal W, Jantarabenjakul W, Wacharachaisurapol N, Tawan M, Puthanakit T, Wittayalertpanya S, et al. Pharmacokinetics of isoniazid and rifapentine in young pediatric patients with latent tuberculosis infection. Int J Infect Dis 2022;122:725–732.

  10. Sakai Y, Fukami T, Nagaoka M, Hirosawa K, Ichida H, Sato R, et al. Arylacetamide deacetylase as a determinant of the hydrolysis and activation of abiraterone acetate in mice and humans. Life Sci 2021;284:119896.

  11. Kobayashi Y, Fukami T, Nakajima A, Watanabe A, Nakajima M, Yokoi T. Species differences in tissue distribution and enzyme activities of arylacetamide deacetylase in human, rat, and mouse. Drug Metab Dispos 2012;40:671–679.

  12. Kurokawa T, Fukami T, Yoshida T, Nakajima M. Arylacetamide deacetylase is responsible for activation of prasugrel in human and dog. Drug Metab Dispos 2016;44:409–416.

  13. Bertelsen B, Stefánsson H, Riff Jensen L, Melchior L, Mol Debes N, Groth C et al (2016) Association of AADAC Deletion and Gilles de la Tourette Syndrome in a Large European Cohort. 79.

  14. Trickett JI, Patel DD, Knight BL, Saggerson ED, Gibbons GF, Pease RJ (2001) Characterization of the rodent genes for arylacetamide deacetylase, a putative microsomal lipase, and evidence for transcriptional regulation. J Biol Chem 276:39522–39532.

    Article  Google Scholar 

  15. Yuan L, Zheng W, Yang Z, Deng X, Song Z, Deng H (2018) Association of the AADAC gene and Tourette syndrome in a Han Chinese cohort. Neurosci Lett 666:24–27.

    Article  Google Scholar 

  16. Pagliaroli L, Vereczkei A, Padmanabhuni SS, Tarnok Z, Farkas L, Nagy P et al (2020) Association of Genetic Variation in the 3’UTR of LHX6, IMMP2L, and AADAC With Tourette Syndrome. Front Neurol 11:803.

    Article  Google Scholar 

  17. Wu C, Wu Z, Tian B (2020) Five gene signatures were identified in the prediction of overall survival in resectable pancreatic cancer. BMC Surg 20:1–10.

    Article  Google Scholar 

  18. Wang Y, Fang T, Wang Y, Yin X, Zhang L, Zhang X et al (2022) Impact of AADAC gene expression on prognosis in patients with Borrmann type III advanced gastric cancer. BMC Cancer 22:1–19.

    Article  Google Scholar 

  19. Abdel-Tawab MS, Fouad H, Othman AM, Eid RA, Mohammed MA, Hassan A et al (2022) Evaluation of gene expression of PLEKHS1, AADAC, and CDKN3 as novel genomic markers in gastric carcinoma. PLoS One 17:e0265184.

    Article  Google Scholar 

  20. Verbruggen B, Gunnarsson L, Kristiansson E, Österlund T, Owen SF, Snape JR et al (2018) ECOdrug: a database connecting drugs and conservation of their targets across species. Nucleic Acids Res 46:D930.

    Article  Google Scholar 

  21. Matsuya A, Sakate R, Kawahara Y, Koyanagi KO, Sato Y, Fujii Y et al (2008) Evola: Ortholog database of all human genes in H-InvDB with manual curation of phylogenetic trees. Nucleic Acids Res 36:D787–D792.

    Article  Google Scholar 

  22. Uno Y, Hosokawa M, Imai T (2015) Isolation and characterization of arylacetamide deacetylase in cynomolgus macaques. J Vet Med Sci 77:721–724.

    Article  Google Scholar 

  23. Honda S, Fukami T, Tsujiguchi T, Zhang Y, Nakano M, Nakajima M (2021) Hydrolase activities of cynomolgus monkey liver microsomes and recombinant CES1, CES2, and AADAC. Eur J Pharm Sci 161:105807.

    Article  Google Scholar 

  24. Yoshida T, Fukami T, Kurokawa T, Gotoh S, Oda A, Nakajima M (2018) Difference in substrate specificity of carboxylesterase and arylacetamide deacetylase between dogs and humans, vol 111. Elsevier B.V.

    Book  Google Scholar 

  25. Jones P, Binns D, Chang HY, Fraser M, Li W, McAnulla C et al (2014) InterProScan 5: Genome-scale protein function classification. Bioinformatics 30:1236–1240.

    Article  Google Scholar 

  26. Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35:1547–1549.

    Article  Google Scholar 

  27. Ciccarelli FD, Doerks T, Von Mering C, Creevey CJ, Snel B, Bork P (2006) Toward automatic reconstruction of a highly resolved tree of life. Science (80- ) 311:1283–1287.

    Article  Google Scholar 

  28. Charif D, Lobry JR (2007) SeqinR 1.0-2: a contributed package to the R project for statistical computing devoted to biological sequences retrieval and analysis. In: Bastolla U, Porto M, Roman HE, Vendruscolo M (eds) Struct. Approaches to Seq. Evol. Mol. Networks, Popul. Springer Berlin Heidelberg, Berlin, Heidelberg, pp 207–232.

    Chapter  Google Scholar 

  29. Szklarczyk D, Gable AL, Lyon D, Junge A, Wyder S, Huerta-Cepas J et al (2019) STRING v11: Protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic Acids Res 47:D607–D613.

    Article  Google Scholar 

  30. Oughtred R, Rust J, Chang C, Breitkreutz BJ, Stark C, Willems A et al (2021) The BioGRID database: a comprehensive biomedical resource of curated protein, genetic, and chemical interactions. Protein Sci 30:187–200.

    Article  Google Scholar 

  31. Tareen A, Kinney JB (2020) Logomaker: beautiful sequence logos in Python. Bioinformatics 36:2272–2274.

    Article  Google Scholar 

  32. Schneider TD, Stephens RM (1990) Sequence logos: a new way to display consensus sequences. Nucleic Acids Res 18:6097–6100.

    Article  Google Scholar 

  33. Bailey TL, Johnson J, Grant CE, Noble WS (2015) The MEME Suite. Nucleic Acids Res 43:W39–W49.

    Article  Google Scholar 

  34. Mirdita M, Schütze K, Moriwaki Y, Heo L, Ovchinnikov S, Steinegger M (2022) ColabFold: making protein folding accessible to all. Nat Methods 196(19):679–682.

    Article  Google Scholar 

  35. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC et al (2004) UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612.

    Article  Google Scholar 

  36. Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD et al (2005) Protein identification and analysis tools on the ExPASy server. Proteomics Protoc Handb:571–607.

  37. Möller S, Croning MDR, Apweiler R (2001) Evaluation of methods for the prediction of membrane spanning regions. Bioinformatics 17:646–653.

    Article  Google Scholar 

  38. Steentoft C, Vakhrushev SY, Joshi HJ, Kong Y, Vester-Christensen MB, Schjoldager KTBG et al (2013) Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology. EMBO J 32:1478–1488.

    Article  Google Scholar 

  39. Williams ET, Ehsani ME, Wang X, Wang H, Qian YW, Wrighton SA et al (2008) Effect of buffer components and carrier solvents on in vitro activity of recombinant human carboxylesterases. J Pharmacol Toxicol Methods 57:138–144.

    Article  Google Scholar 

  40. Penning TM (1993) Dihydrodiol dehydrogenase and its role in polycyclic aromatic hydrocarbon metabolism. Chem Biol Interact 89:1–34.

    Article  Google Scholar 

  41. Gilissen J, Jouret F, Pirotte B, Hanson J (2016) Insight into SUCNR1 (GPR91) structure and function. Pharmacol Ther 159:56–65.

    Article  Google Scholar 

  42. Wang J, Molday LL, Hii T, Coleman JA, Wen T, Andersen JP et al (2018) Proteomic analysis and functional characterization of P4-ATPase phospholipid flippases from murine tissues. Sci Rep 8:1–14.

    Article  Google Scholar 

  43. Sood S, Sharma A, Sharma N, Kanwar SS (2018) Carboxylesterases: sources, characterization and broader applications. Insights Enzym Res 01.

  44. Laurell H, Contreras JA, Castan I, Langin D, Holm C (2000) Analysis of the psychrotolerant property of hormone-sensitive lipase through site-directed mutagenesis. Protein Eng 13:711–717.

    Article  Google Scholar 

  45. Higa M, Fujita M, Yoshida K (2017) DNA replication origins and fork progression at mammalian telomeres. Genes 8:112.

    Article  Google Scholar 

  46. Martínez P, Blasco MA (2018) Heart-breaking telomeres. Circ Res 123:787–802.

    Article  Google Scholar 

  47. De Lange T (2018) Shelterin-mediated telomere protection. Annu Rev Genet 52:223–247.

    Article  Google Scholar 

  48. Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132.

    Article  Google Scholar 

  49. Guruprasad K, Reddy BVB, Pandit MW (1990) Correlation between stability of a protein and its dipeptide composition: a novel approach for predicting in vivo stability of a protein from its primary sequence. Protein Eng Des Sel 4:155–161.

    Article  Google Scholar 

  50. Watanabe A, Fukami T, Takahashi S, Kobayashi Y, Nakagawa N, Nakajima M et al (2010) Arylacetamide deacetylase is a determinant enzyme for the difference in hydrolase activities of phenacetin and acetaminophen. Drug Metab Dispos 38:1532–1537.

    Article  Google Scholar 

  51. Mizoi K, Takahashi M, Sakai S, Ogihara T, Haba M, Hosokawa M (2020) Structure-activity relationship of atorvastatin derivatives for metabolic activation by hydrolases. Xenobiotica 50:261–269.

    Article  Google Scholar 

  52. Muta K, Fukami T, Nakajima M, Yokoi T (2014) N-Glycosylation during translation is essential for human arylacetamide deacetylase enzyme activity. Biochem Pharmacol 87:352–359.

    Article  Google Scholar 

  53. An HJ, Froehlich JW, Lebrilla CB (2009) Determination of glycosylation sites and site-specific heterogeneity in glycoproteins. Curr Opin Chem Biol 13:421–426.

    Article  Google Scholar 

  54. Frick C, Atanasov AG, Arnold P, Ozols J, Odermatt A (2004) Appropriate function of 11β-hydroxysteroid dehydrogenase type 1 in the endoplasmic reticulum lumen is dependent on its N-terminal region sharing similar topological determinants with 50-kDa esterase. J Biol Chem 279:31131–31138.

    Article  Google Scholar 

  55. Konishi K, Fukami T, Ogiso T, Nakajima M (2018) In vitro approach to elucidate the relevance of carboxylesterase 2 and N-acetyltransferase 2 to flupirtine-induced liver injury. Biochem Pharmacol 155:242–251.

    Article  Google Scholar 

Download references


The authors would like to thank Dr. Mariana Díaz-Tenorio for her kind advice and helpful discussions.


This study was supported by PROFAPI-ITSON grant (DFP300320165). We gratefully recognize a postdoctoral fellowship for T.D.-V. from SEP-SES (23-007-C) postdoctoral grants. 

Author information

Authors and Affiliations



All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by TD-V, CBR-O, and RBM-P. The first draft of the manuscript was written by TD-V, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript

Corresponding author

Correspondence to Raúl Balam Martínez-Pérez.

Ethics declarations

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1: Figure S1.

Amino acid sequence alignments for AADAC orthologues. The name of the orthologues is shown as an accession number on the left side. "⁎" shows identical residues for AADAC orthologues; ": "2 alternate residues. On grey color, principal identical residues referent to HGGG box, Consensus sequence (GXSXG), and YXLXP motif. Loops above the amino acid sequence indicate alpha-helix structures, and arrows indicate beta-sheet structures. Figure S2. The complete motifs analysis, using MEME-suite of AADAC orthologues. Table S1. Summary of the physicochemical characterization, transmembrane regions, and N-glycosylation sites predicted for AADAC orthologue. Table S2. The complete protein-protein interaction network analysis of AADAC orthologues using the STRING tool.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Diaz-Vidal, T., Romero-Olivas, C.B. & Martínez-Pérez, R.B. Characterization, comparative, and functional analysis of arylacetamide deacetylase from Gnathostomata organisms. J Genet Eng Biotechnol 20, 169 (2022).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: