Eis, a novel family of arylalkylamine N-acetyltransferase (EC 2.3.1.87)

Enhanced intracellular survival (Eis) proteins were found to enhance the intracellular survival of mycobacteria in macrophages by acetylating aminoglycoside antibiotics to confer resistance to these antibiotics and by acetylating DUSP16/MPK-7 to suppress host innate immune defenses. Eis homologs composing of two GCN5 N-acetyltransferase regions and a sterol carrier protein fold are found widely in gram-positive bacteria. In this study, we found that Eis proteins have an unprecedented ability to acetylate many arylalkylamines, are a novel type of arylalkylamine N-acetyltransferase AANAT (EC 2.3.1.87). Sequence alignment and phyletic distribution analysis confirmed Eis belongs to a new aaNAT-like cluster. Among the cluster, we studied three typical Eis proteins: Eis_Mtb from Mycobacterium tuberculosis, Eis_Msm from Mycobacterium smegmatis, and Eis_Sen from Saccharopolyspora erythraea. Eis_Mtb prefers to acetylate histamine and octopamine, while Eis_Msm uses tyramine and octopamine as substrates. Unlike them, Eis_Sen exihibits good catalytic efficiencies for most tested arylalkylamines. Considering arylalkylamines such as histamine plays a fundamental role in immune reactions, future work linking of AANAT activity of Eis proteins to their physiological function will broaden our understanding of gram-positive pathogen-host interactions. These findings shed insights into the molecular mechanism of Eis, and reveal potential clinical implications for many gram-positive pathogens.

physiological functions the nervous, neuroendocrine and immune systems, including histamine, dopamine, octopamine, tyramine, tryptamine, norepinephrine, methoxytryptamine, serotonin, and 5-hydroxytryptamine 9 . The acetylation of arylalkylamines by arylalkylamine N-acetyltransferase (AANAT) is crucial for the maintenance of normal physiological functions. For example, N-acetylation of 5-hydroxytryptamine by AANAT is a rate-limiting step for the synthesis of melatonin in vertebrates. AANAT plays a unique role in vertebrate biology by controlling the rhythmic production of melatonin in the pineal gland. Hence, AANAT has been referred to as the "Timezyme" 10 . In insects, aaNATs also play important roles such as the cuticle sclerotization and the inactivation of monoamine neurotransmitters 11 .
In this study, we characterized three Eis N-acetyltransferase Eis_Mtb, Eis_Msm, and Eis_Sen. Interestingly, Eis proteins are all capable of acetylating arylalkylamines (histamine, octopamine, tyramine, octopamine, etc) but with different substrates specificity. The results indicated that Eis are a novel family of arylalkylamine N-acetyltransferase (EC 2.3.1.87). Eis proteins are found widely in gram-positive bacteria, including many pathogens such as Mycobacterium, Enterococcus, Bacillus, Listeria, and Clostridium spp. Our findings indicate that Eis may play a role in gram-positive pathogen-host interactions, which may reveal significant clinical implications.

Results and Discussion
Eis enzymes are found mostly in Gram-positive bacteria. Eis proteins contain N-terminal GNAT, central GNAT, and C-terminal SCP2 domains. The name "Eis" originates from the enhanced intracellular survival protein Eis of M. tuberculosis. The InterPro database (v48.0) comprises a total of 5152 proteins containing the Eis domain (http://www.ebi.ac.uk/interpro/entry/IPR025559). Most of these proteins (91.6%) are found in Gram-positive bacteria (2337 proteins in Firmicutes and 2380 proteins in Actinobacteria). Among 2369 Actinomycetales Eis proteins, 1868 proteins are found in Mycobacteria. As shown in Table 1, in Firmicutes, Eis proteins are found mostly in Bacillales (357), Lactobacillales (1460), and Clostridiales (434). Many pathogens, including Mycobacterium, Enterococcus, Bacillus, Listeria, and Clostridium spp., harbor Eis domain-containing proteins. Remarkably, most of the Eis domain (99%) is fused to another N-terminal GNAT domain to generate Eis enzymes with a unique domain organization (GNAT-Eis or GNAT-GNAT-SCP2), and exhibiting AG-acetylating acetyltransferase activity.

Exploration of arylalkylamines as potential substrates of Eis. Previous studies have shown that
Eis_Mtb is capable of deactivation the various anti-TB drugs via acetylation. Anti-TB drugs such as AGs, isoniazid, pyrazinamide, ciprofloxacin and other substrates of Eis_Mtb including lysine, di-lysine, tri-lysine, tetra-lysine, lisinopril, tuftsin, thymopoietin II, and peptide fragment, all possess the same part in their molecular structure, that's the free amino group 8 . Based on this, we tried to explore whether Eis_Mtb could acetylate another major class of molecular containing the free amino group, arylalkylamines. To investigate whether Eis acetylates the arylalkylamines, the acetylation activities and kinetic parameters of Eis_Mtb, Eis_Msm, and Eis_Sen were determined for several arylalkylamines, including dopamine, tyramine, octopamine, serotonin, histamine, typtamine and phenethylamine. A typical arylalkylamine N-acetyltransferase (EC 2.3.1.87) aaNAT2 from Aedes aegypti was used as a control. The results about apparent kinetic constants are shown in Table 2. Eis_Mtb exihibits catalytic activity towards histamine and octopamine. Eis_Msm could not acetylate most of arylalkylamines, but exhibits affinity to octopamine and tyramine even with a high Km value. Interestingly, Eis_Sen showed a broad substrate specificity toward most of arylalkylamines, and exhibits the highest affinity to octopamine. The K m value of Eis_Mtb is higher comparative to the aaNAT2 from Aedes aegypti but the catalytic efficiency (k cat /K m ) of Eis_Mtb is lower, indicating Eis_Mtb has lower affinity and catalytic efficiency for arylalkylamines. However, not all of the identified aaNAT have good kinetic constants towards arylalkylamines. For example, an AANAT from Saccharomyces cerevisiae (scAANAT) was studied and its specific activity was also very low. The K m values of scAANAT for serotonin was 6.5 mM and for phenylethylamine was as much as 13.3 mM (with Ac-CoA = 0.5 mM) 12 . In addition, Falcon and his co-workers characterized nonvertebrate AANAT from C. milii, and its K m value for tyramine and serotonin were as much as 44.87 mM and 59.46 mM respectively 13 .
To further examine substrate specificities of Eis proteins, various acyl-CoA substrates with increasing acyl chain lengths were investigated at saturating amine substrate with the highest affinity ( Table 3). The results showed that aaNAT2 revealed the broad specificities of the acyl-CoA substrates. Eis can only exhibits activity to short chain acyl-CoA substrates, acetyl-CoA and propionyl-CoA. No activities for butyryl-CoA and octanoyl-CoA was observed. A structural difference between the active sites of Eis and aaNAT2 might contribute to their substrate specificities that Eis enzymes are inactive for acyl-CoA substrates with the longer acyl chain lengths (>C3). We found a similar trend between aaNAT2 and Eis was that the K m values and the relative (k cat / K m ) values did decrease when increasing the acyl chain length of acyl-CoA substrates ( Table 3). The decrease of k cat values significantly resulted in the decline of the second-order rate. This phenomenon was also observed in aaNATL7 14,15 . The long-chain acyl-CoA substrates might perturb or block the amine substrate binding because of their extension into the binding pocket of amine substrate in Eis enzymes or aaNATL7.
aaNAT2 from Aedes aegypti Tyramine (20 ± 4.0) × 10 2 11 ± 0.8 (5.7 ± 1.5) × 10 3 Octopamine   Arylalkylamine N-acetyltransferases usually belong to bisubstrate enzymes. The substrate binding order of multi-substrate enzymes is important to understand their catalytic mechanism. The previous researches demonstrated that D. melanogaster AANATA and sheep serotonin N-acetyltransferase both revealed an ordered sequential kinetic mechanism 15,16 . To scrutinize and compare the catalytic mechanism of Eis and aaNAT enzymes, we investigated the order of acetyl-CoA and arylalkylamine to bind to Eis using the dead-end inhibition experiments. Oleoyl-CoA (an analogue of acetyl-CoA) and tyrosol (a structural analogue of the amine substrate) were used as the inhibitors 14,15 . We found that oleoyl-CoA was competitive versus acetyl-CoA and noncompetitive versus histamine, with inhibition constants of 268 ± 68 nM and 509 ± 94 nM, respectively (Fig. 1). Tyrosol was uncompetitive versus acetyl-CoA and competitive versus histamine, with inhibition constants of 130 ± 18 μM and 175 ± 49 μM, respectively. These observations indicated that Eis enzymes showed an ordered sequential mechanism, similar to the catalytic mechanism of aaNAT 15,16 . N-acetylhistamine was produced with acetyl-CoA binding first followed by histamine to form a precatalytic ternary eis·acetyl-CoA·histamine complex. It is worth noting that the inhibition results ( Fig. 1) are consistent with an ordered kinetic mechanism, but that other kinetic mechanisms are also possible.

Evolution analysis of Eis enzymes and other studied aaNATs.
To understand the phylogenetic relationship between Eis and other known aaNATs, we constructed a phylogenetic tree (Fig. 2). In this study, most of aaNATs chosen on the phylogenetic tree are based on previous reports 17 . The sequences of GNAT domains in Eis enzymes were used for phylogenetic analysis. Phyletic distribution analysis confirmed four major clusters, termed clusters 1 (orange), 2 (cyan), 3 (blue), and 4 (green). Cluster 1 represent typical insect aaNATs, all identified insect aaNATs are located in cluster 1. The aaNAT-2 in cluster 1 was selected as a control. Mosquito aaNATs were clustered into two areas (cyan and blue). We found that all Eis homologues could be clustered into the one clade, including the Eis_Mtb, Eis_Msm, and Eis_Sen proteins from this set.  Table 4. Despite multiple sequence alignment of N-acyltransferase superfamily region showing low pairwise sequence identity (Fig. 3A), superimposition of the structures of N-acyltransferase superfamily within Eis_Mtb, Eis_Msm, Eis_Sen, and aaNAT revealed that Eis enzymes was very similar to aaNATs (Fig. 3B-E). Superimposition of the N-acyltransferase superfamily structure of Eis_Mtb with the structure of aaNAT2 showed that r.m.s. deviation was 4.636 Å for 33 Cα (Fig. 3B). N-acyltransferase superfamily region in Eis_Mtb increased by 25 amino acids compared with that of aaNAT2, so new superimposition of the 71-119 sequences of the N-acyltransferase superfamily structure in Eis_Mtb with the structure in aaNAT2 showed that r.m.s. deviation was 3.724 Å for 43 Cα (Fig. 3C) after deleting redundant amino acids. Superimposition of the N-acyltransferase superfamily structure of Eis_Msm with the structures of aaNAT2 showed that r.m.s. deviation was 2.355 Å for 46 Cα (Fig. 3D). The consequences of superimposition analysis illustrated that N-acyltransferase superfamily structure in Eis enzymes is very similar to the structure of aaNAT2 especially Eis_Msm is the most similar because of their the lowest r.m.s. deviation value, which further indicated that Eis enzymes are capable of acetylating arylalkylamines as well as aaNATs.
Our study revealed that Eis enzyme was a novel family of arylalkylamine N-acetyltransferase (aaNAT, EC 2.3.1.87) catalyzing the generation of N-acylarylalkylamides from the short chain acyl-CoA and a broad array of corresponding arylalkylamine substrates, although the catalytic efficiency was lower than that of the typical aaNATs. As well as aaNAT2, Eis was less discriminatory for the amine substrates including dopamine, histamine, tyramine, octopamine, phenethylamine, tryptamine, and serotonin. Actually, Eis was more discriminative for the acyl-CoA substrates, only showing activity to acetyl-CoA and propionyl-CoA and the k cat /K m values for acetyl-CoA is ≥3-fold higher than that of propionyl-CoA. Eis enzymes showed an ordered sequential mechanism, similar to the catalytic mechanism of typical aaNAT 15,16 . However an arylamine N-Acetyltransferase from Mycobacterium tuberculosis revealed the bi-bi ping-pong kinetic mechanism 18 . In addition, the evolution analysis and structural comparison indicated that Eis is capable of acetylating arylalkylamines, as a novel family of arylalkylamine N-acetyltransferase. Considering arylalkylamines such as histamine plays a fundamental role in immune reactions 19 , future work linking of AANAT activity of Eis proteins to their physiological function will broaden our understanding of gram-positive pathogen-host interactions. These findings shed insights into the molecular mechanism of Eis and reveal potential clinical implications for many gram-positive pathogens.

Materials and Methods
Bacterial strains and reagents. S   within Eis_Mtb (colored in red) onto the structure within aaNAT2 (colored in yellow). (C) Superimposition of the 71-119 sequences of N-acyltransferase superfamily structure within Eis_Mtb (colored in red) onto the structure within aaNAT2 (colored in yellow). (D) Superimposition of the N-acyltransferase superfamily structure within Eis_Msm (colored in red) onto the structure within aaNAT2 (colored in yellow). (E) Superimposition of the 71-119 sequences of the N-acyltransferase superfamily structure within Eis_Mtb (colored in red) and the structure within Eis_Msm (colored in blue) onto the structure within aaNAT2 (colored in yellow).