Structural and Mechanistic Analysis of Drosophila melanogaster Agmatine N-Acetyltransferase, an Enzyme that Catalyzes the Formation of N-Acetylagmatine

Agmatine N-acetyltransferase (AgmNAT) catalyzes the formation of N-acetylagmatine from acetyl-CoA and agmatine. Herein, we provide evidence that Drosophila melanogaster AgmNAT (CG15766) catalyzes the formation of N-acetylagmatine using an ordered sequential mechanism; acetyl-CoA binds prior to agmatine to generate an AgmNAT•acetyl-CoA•agmatine ternary complex prior to catalysis. Additionally, we solved a crystal structure for the apo form of AgmNAT with an atomic resolution of 2.3 Å, which points towards specific amino acids that may function in catalysis or active site formation. Using the crystal structure, primary sequence alignment, pH-activity profiles, and site-directed mutagenesis, we evaluated a series of active site amino acids in order to assign their functional roles in AgmNAT. More specifically, pH-activity profiles identified at least one catalytically important, ionizable group with an apparent pKa of ~7.5, which corresponds to the general base in catalysis, Glu-34. Moreover, these data led to a proposed chemical mechanism, which is consistent with the structure and our biochemical analysis of AgmNAT.


Results and Discussion
Crystal structure of AgmNAT. A homology model for AgmNAT was constructed using the Aedes aegypti arylalkylamine N-acetyltransferase structure 21 as a template for molecular replacement. The AgmNAT (CG15766) crystal structure was determined at 2.3Å, with two monomers in the asymmetric unit of the P2 1 space group ( Table 1). The two monomers are nearly identical with an RMSD value of 0.262 Å when aligning 862 backbone atoms. Similar to the arylalkylamine N-acetyltransferase model, the new structure is primarily composed of six α-helices and seven anti-parallel α-strands (Fig. 1A). The AgmNAT structure displays a conserved GNAT fold, similar to that observed for D. melanogaster AANATA and human spermidine/spermine N 1 -acetyltransferase (SSAT) ( Supplementary Fig. S2), though the sequence identity is low when compared to these N-acetyltransferase enzymes (24% with AANATA and <20% for SSAT), a known feature of GNAT enzymes 2 . Based on the functional and structural similarities between AgmNAT and other GNATs such as AANATA (PDB 3TE4) 15,60 , we predict the active site pocket to be similar, though not identical, for the binding of the acyl-CoA and amine substrates (Fig. 2). The active site is well defined in the 2Fo-Fc electron density map (Fig. 1B,C) and is located near the crystal packing interface for both monomers. Based on the structure of AANATA with acetyl-CoA bound (PDB 3TE4) 60 , the binding surface for the adenosine 3-phosphate 5-pyrophosphate moiety of CoA-SH is blocked by protein-protein interactions in the AgmNAT structure, but the rest of the active site is open. The splaying of β-strand four and five, a conserved structural feature in GNAT enzymes, is also displayed in AgmNAT, which is the binding site for the pantetheine arm of acetyl-CoA 2 . Moreover, a conserved glutamate, Glu-34, that serves as the catalytic base for other D. melanogaster N-acyltransferase enzymes, is located within an accessible pocket that can accommodate the acyl-CoA and amine substrate, similar to that observed for AANATA (Fig. 1B) 15 . Also observed in the active site pocket are the residues, Pro-35 and Ser-171 (Fig. 1B,C), which are conserved amino acids that regulate catalysis in other D. melanogaster N-acyltransferases 15,61,62 . The functional roles of Pro-35 and Ser-171 of AgmNAT are discussed in subsequent sections.
Evaluation of acyl-CoA steady-state kinetic constants. AgmNAT showed minimal differences in the measured K m,app values for acyl-CoA substrates ranging from acetyl-CoA to decanoyl-CoA (C2-C10) ( Table 2) when agmatine was used as the saturating amine substrate. However, there was an acyl chain length dependent decrease in the apparent k cat value for the acyl-CoA substrates as the chain length is increased. This apparent decrease in the turnover number of ~150-fold from acetyl-CoA to decanoyl-CoA, led to the observed acyl-chain length specific decrease in the (k cat /K m ) app value. In addition, oleoyl-CoA was not a substrate at a concentration of 500 μM. These data likely result from the acyl-chain partially (decanoyl-CoA) or fully (oleoyl-CoA) occupying the amine binding site, perturbing the productive binding of agmatine; therefore, resulting in a decrease in or complete loss of catalysis. Similar results were observed for other D. melanogaster N-acyltransferases 15,61,62 . Evaluation of amine substrate steady-state kinetic constants. We screened >50 amines as potential AgmNAT substrates using acetyl-CoA or oleoyl-CoA as the co-substrate because of our interests in fatty acid biosynthesis, structure function relationships of GNAT enzymes, and the development of novel insecticides targeted to this class of enzymes. Our amine substrate screen included the canonical amino acids (except for Cys because Cys reacts with DTNB), amino acid analogs, other biogenic amines, and different xenobiotic amines. Only six amines (Table 3) showed AgmNAT activity >3-fold higher than the level of background acetyl-CoA thioesterase activity, whereas none showed a greater rate for oleoyl-CoA. Also, we identified five polyamines as AgmNAT substrates: spermine, N 8 -acetylspermidine, putrescine, spermidine, and cadaverine ( Table 3). The (k cat /K m ) app values for the polyamines were lower than that measured for agmatine, the (k cat /K m ) app,agmatine /(k cat / K m ) app,polyamine ratio ranging from 15 for spermine to 1900 for cadaverine. Structural evidence for the specificity for agmatine and different polyamines likely results from the acidic nature of the active site, similar to that observed for the human ortholog (human SSAT) (Fig. 3) 2 . A more acidic active site can accommodate an amine substrate with a basic guanidinium group better than one with a hydrophobic aromatic group, giving rise to the difference in substrate specificity when compared to an AANAT 15,60 . AgmNAT was originally named AANATL8 based on primary sequence similarity 15 ; however, the substrate specificity data reported here support a new designation: agmatine N-acetyltransferase. This is the first report of agmatine serving as the best amine substrate for an N-acyltransferase. There are only a few reports of agmatine serving as a substrate within this family of enzymes 17,62,63 and only two reports on the identification of N-acetylagmatine from a biological source 64,65 . Rats fed heavy-atom labeled agmatine yielded two major urinary products; heavy-atom labeled N-acetylagmatine and unprocessed, but labeled agmatine 64 , suggesting a similar conversion as that catalyzed by AgmNAT. Inactivation of agmatine neurotransmission by N-acetylation is an underappreciated reaction between arginine, agmatine, and human disease 27,66-68 , the search for a human ortholog of Drosophila AgmNAT could lead to a new target for drug development. Additionally, selective targeting of Drosophila AgmNAT could result in the development of novel insecticides for insect control [20][21][22][23] . We found that arginine, arginine methyl ester, N-acetylputrescine, and N 1 -acetylspermidine were not AgmNAT substrates. The ~25-fold increase in k cat,app for N 8 -acetylspermidine when compared to spermidine, together with our data demonstrating that N-acetylputrescine and N 1 -acetylspermidine were not substrates all suggest that AgmNAT, most likely, catalyzes the mono-and N1-specific acetylation of these biogenic amines, similar to what is observed for the mammalian spermidine N-acetyltransferase 69,70 .  The increase in the k cat,app value, together with the small ~2-fold difference in the K m,app for N 8 -acetylspermidine relative to spermidine, could result from non-productive binding of the N8-amine of spermidine in the AgmNAT active site, whereby the N1-amine is better positioned for catalysis: deprotonation and then nucleophilic attack of the -NH 2 at the carbonyl of the acetyl-CoA thioester moiety. This means both of the amine moieties can bind in the active site, but only the N1-amine is acetylated.
While arginine and arginine methyl ester are not AgmNAT substrates, we further evaluated these for AgmNAT inhibition to determine if either could bind to the enzyme. Arginine methyl ester proved to weakly inhibit AgmNAT, decreasing the rate of N-acetylagmatine formation from acetyl-CoA and agmatine by ~50% at 10 mM. In contrast, we found no inhibition of N-acetylagmatine formation at both 10 mM and 25 mM arginine. These data show that a modification of the α-position of agmatine inhibits binding to AgmNAT and that the inhibition results from both electronic and steric effects. The presence of the negatively charged α-carboxylate seems to eliminate or significantly weaken AgmNAT binding, likely the result of charge-charge repulsion. Evidence for this suggestion comes from the weak inhibition by arginine methyl ester (K i,s and K i,i ≥ 1 mM, Supplementary  Fig. S3), but no apparent inhibition by arginine at a concentration as high as 25 mM.
( Fig. 4B). These data suggest that the AgmNAT-catalyzed formation of N-acetylagmatine occurs via a sequential mechanism; catalysis takes place only after formation of the AgmNAT•acetyl-CoA•agmatine ternary complex. Next, we determined if the AgmNAT kinetic mechanism is an ordered or random sequential mechanism by using substrate analogs, oleoyl-CoA, arcaine, and arginine methyl ester, as dead-end inhibitors vs. acetyl-CoA and agmatine. The inhibitor data is summarized in Table 4 and we have included the double reciprocal plots for the inhibitors in the Supplementary Materials. Arcaine is structurally related to agmatine, with its primary amine moiety replaced with a guanidinium group. Arcaine serving as an AgmNAT inhibitor supports our conclusion that AgmNAT does not acetylate the guanidinium amine of agmatine. None of these inhibitors showed any rate of catalysis above the slow, background rate of acetyl-CoA or oleoyl-CoA hydrolysis. Oleoyl-CoA produced competitive and noncompetitive inhibition plots for acetyl-CoA and agmatine (Table 4 and Supplementary Fig. S4A,B). Arcaine produced uncompetitive and competitive inhibition plots for acetyl-CoA and agmatine (Table 4 and Supplementary Fig. S4C,D). As observed for arcaine, arginine methyl ester produced uncompetitive and competitive inhibition plots for acetyl-CoA and agmatine (Table 4 and Supplementary Fig. S3). These data demonstrate that AgmNAT catalyzes the formation of N-acetylagmatine through an ordered sequential mechanism: acetyl-CoA binding first followed by agmatine to generate the AgmNAT•acetyl-CoA•agmatine complex prior to catalysis. This is similar to the kinetic mechanism for other D. melanogaster GNAT enzymes, including AANATA, AANATL2, and AANATL7 [15][16][17] . Support for ordered sequential mechanism for AgmNAT comes from a statistically better fit to Equation 3 (as shown in Fig. 4) and the noncompetitive inhibition of oleoyl-CoA vs. agmatine (Table 4 and Supplementary  Fig. S4A,B). Additional support and further details for the kinetic mechanism are revealed by N-acetylagmatine product inhibition. N-Acetylagmatine produced uncompetitive and competitive inhibition plots for acetyl-CoA and agmatine (Table 4 and Supplementary Fig. S5). Uncompetitive inhibition by N-acetylagmatine vs. acetyl-CoA ( Supplementary Fig. S5A) is inconsistent with a ping pong kinetic mechanism. In sum, the kinetic analyses are consistent with two kinetic mechanisms: (a) ordered sequential substrate binding with acetyl-CoA binding first followed by ordered sequential product release with N-acetylagmatine being released last or (b) ordered sequential substrate binding with acetyl-CoA binding first followed by ordered sequential product release with CoA-SH being being released last. Uncompetitive inhibition by N-acetylagmatine vs. acetyl-CoA would be explained by the formation of a non-productive AgmNAT•acetyl-CoA•N-acetylagmatine complex with no reversible connection between the AgmNAT•acetyl-CoA complex and the AgmNAT•CoA-SH complex. We favor the latter mechanism because we have demonstrated that CoA-SH will bind to other D. melanogaster AANATs 15,61 and many other N-acetyltransferases exhibit ordered product release with CoA-SH being released last 71-74 . Proposed AgmNAT chemical mechanism. We combined the pH-dependence of the kinetic constants, primary sequence alignment to other D. melanogaster GNAT enzymes 15 , determination of three-dimensional structure, and site-directed mutagenesis of a putative catalytically important residue to provide insights into the AgmNAT chemical mechanism. First, the pH-dependence of the kinetic constants was assessed for acetyl-CoA to assign apparent pK a values to ionizable groups involved in catalysis. Both the k cat,app and (k cat /K m ) app pH-rate profiles produced a rising profile with a pK a,app of 7.7 ± 0.1 and 7.3 ± 0.2, respectively (Fig. 5). An apparent pK a of ~7.5 can be attributed to a general base in catalysis, likely either deprotonation of the primary amine of agmatine or the zwitterionic tetrahedral intermediate generated upon nucleophilic attack of agmatine at the carbonyl thioester of acetyl-CoA. A second, higher pK a,app , possibly resulting from the deprotonation of a catalytically important general acid, was not observed in our pH-activity data, a surprising result given that a pK a ~8.5-9.5 has been observed for many other N-acyltransferases 2,75,76 . Explanations for these data include: (a) AgmNAT catalysis does not require a general acid, (b) the general acid in catalysis is not rate-limiting under our assay conditions, or (c) the general acid in AgmNAT catalysis has an apparent pK a > 9.5. Because of the high rate of base-catalyzed acyl-CoA hydrolysis, we cannot perform experiments at pH > 9.5 to define a pK a > 9.5.
Next, we combined information from primary sequence alignments, the AgmNAT structure, and site-directed mutagenesis to define potential amino acids that could function in catalysis. A conserved glutamate has been proposed as the catalytic base in two D. melanogaster arylalkylamine N-acetyltransferases (AANATs), which corresponds to Glu-34 in AgmNAT 15,16 . Additionally, the AgmNAT structure shows that Glu-34 is in the active site, a buried region with several structural waters positioned within proximity of Glu-34 (Fig. 1B), similar to D. melanogaster AANATA (PDB code: 3TE4) 15 . Ordered water molecules within the active site of other GNAT enzymes are thought to form a "proton wire" that assists the general base in catalysis 2,15,17,63,[75][76][77] . Although only a number of water molecules (36 in total) were sufficiently ordered to be modeled in the current structure, the majority of them are in the active sites of the two monomers. The closest ordered water molecules to Glu-34 is ~ 3.7 Å from the Oε 1 , positioned slightly too far for a hydrogen bond; however, we anticipate that the conformational changes upon substrate binding could promote hydrogen bond interactions between ordered water molecules and the functional groups in AgmNAT and substrate. Such hydrogen bonds could facilitate proton transfer from the amine substrate to initiate catalysis. In addition, unlike Glu-33, which is exposed to the bulk solvent, Glu-34 is relatively sheltered and placed close to the hydrophobic core of the protein and next to residues such as Leu-36. This microenvironment could be responsible for a pK a shift of Glu-34, as that identified in the pH-rate profiles. Therefore, we sought to interrogate the catalytic role of Glu-34 by evaluating the kinetic constants of the E34A mutant. The E34A mutation produced a catalytically deficient enzyme, exhibiting only 0.05-0.07% of the wildtype k cat,app value indicating that Glu-34 does function in the catalytic cycle. Furthermore, Glu-34 seems to have a role in substrate binding because the K m,app values for both agmatine and acetyl-CoA for the E34A mutant differ from wildtype values, the K m,app for agmatine increases 20-fold and the K m,app for acetyl-CoA decreases 6-fold ( Table 5). The data generated for the E34A mutant is consistent, but does not prove, that Glu-34 serves as the general base in AgmNAT catalysis. To further investigate the role of Glu-34 in catalysis, we generated pH-activity profiles for the E34A mutant (Fig. 6). The k cat,app profile produced a pH-dependent linear increase with slope of 0.7 and (k cat / K m ) app profile with no slope. Attempts to titrate the pH < 8.0 were unsuccessful, by which a rate of CoA-SH release was not observed above the background hydrolysis rate. The linear profile in both the k cat,app and (k cat /K m ) app pH profiles, combined with the deficiency in catalytic rate suggest that Glu-34 serves as the general base in catalysis.
Our steady-state kinetic data identified an ordered sequential mechanism with acetyl-CoA binding first, followed by agmatine to generate the AgmNAT•acetyl-CoA•agmatine ternary complex prior to catalysis. After the ternary complex formation, Glu-34 functions as the general base to deprotonate the positively charged amine  moiety of agmatine, most likely involving a "proton wire" of ordered water molecules, followed by nucleophilic attack of the carbonyl of the acetyl-CoA thioester to generate a zwitterionic tetrahedral intermediate. Breakdown of the intermediate ensues by the departure of coenzyme A, which is, most likely, protonated by the positively charged amine of the intermediate (Fig. 7). This mechanism is consistent with other proposed chemical mechanisms for the N-acyltransferases of D. melanogaster and other organisms 15,16,24,78 . Other amino acids in AgmNAT that function in substrate binding and modulating catalysis. In addition to Glu-34, three other amino acids were individually mutated to alanine to define their function. These residues, Pro-35, Ser-171, and His-206, are conserved between D. melanogaster GNAT enzymes 15 and are proposed to function in active site formation, substrate binding, and/or regulation of catalysis 16,17 . The P35A mutant is catalytically deficient, with a k cat,app value that is ~2% of wildtype, while exhibiting only minimal K m,app differences when compared to wildtype for both acetyl-CoA and agmatine (Table 5). Similar results were observed for the corresponding proline in other GNAT enzymes, except most exhibited a significant K m increase for the corresponding amine, suggesting a role in substrate binding. Furthermore, the structure of sheep serotonin N-acetyltransferase (PDB code: 1CJW), co-crystalized with the tryptamine-acetyl-CoA bisubstrate inhibitor, shows that the corresponding Pro-64 interacts with this inhibitor via a CH-π interaction with the negatively charged face of the aromatic tryptamine moiety 77,79 . Agmatine lacks an aromatic moiety; thus, the Pro-35 of AgmNAT cannot form a CH-π interaction with agmatine, which we propose is the reason for no K m effect for the P35A mutant.   observed for other GNAT enzymes [15][16][17]79 . In the current AgmNAT structure, Pro-35 is stacked on top of the imidazole ring of His-206 side chain (Fig. 2). The extensive van der Waals interaction may make significant contributions to particular active site configurations. Another active site residue evaluated for its role in substrate binding and catalysis is Ser-171. The S171A mutant only retained ~9% of the wildtype k cat,app and also showed a 3-to 4-fold change in the K m,app values for the substrates (a decrease in the K m,app for acetyl-CoA and an increase in the K m,app for agmatine) ( Table 5). The decrease in the k cat,app could be interpreted that Ser-171 functions as a general acid in catalysis to protonate CoA-Sas it leaves the AgmNAT active site. For Ser-171 to function as a general acid during catalysis, the pK a of the serine hydroxyl would have to decrease by ~3-5 pH units to protonate the thiolate anion of the CoA product. We did not observe an apparent pK a in the pH-rate profiles that would correspond to a general acid, arguing against Ser-171 serving in this role. Alternatively, Ser-171 could have an important role in organizing the active site architecture to accommodate both substrates to enable efficient catalysis. Ser-171 is located in the active site, where its Oγ side chain atom forms hydrogen bonds with the backbone oxygen and nitrogen atoms of Ser-168, and a water-mediated interaction with the Thr-167 backbone nitrogen atom, suggesting that the 165-169 strand region in addition to Ser-171 is important in stabilizing the active site pocket to accommodate both substrates and allow for efficient catalysis to occur (Fig. 1C).
The H206A mutant resulted in a k cat,app value that is ~18-fold lower than the wild-type value, whereas the K m,app-acetyl-CoA and K m,app-agmatine increased 2.3-fold and 1.4-fold, respectively. The corresponding residue (His-220) in D. melanogaster AANATA 15 was shown to interact with Tyr-185 and Pro-48 to form part of the active site, an interaction potentially resulting from a conformational change driven by acetyl-CoA binding. We assign a similar function for His-206 in AgmNAT since its general location in the active site is similar to His-220 in D. melanogaster AANATA, and the van der Waals interaction with Pro-35, as described above, is conserved (Fig. 2B). In addition, the His-206 side chain is in van der Waals contact with Ser-168 Cα and Tyr-188 Cε2, as well as several local prolines, Pro-203 and Pro-205. This means that His-206 is contributing to the formation of the active site by interacting with multiple residues. The apo-AgmNAT structure shows Tyr-170 in a position that is not optimal for a direct interaction with His-206 ( Fig. 2A), unlike that shown for the corresponding residues in the AANATA structure co-crystalized with acetyl-CoA 15,60 . Tyr-170 occupies space near the entry point for acetyl-CoA into its binding pocket; therefore, we predict that a conformational change will occur that will move Tyr-170 into position for optimal acetyl-CoA binding, possibly by interacting with His-206.
The findings presented in this manuscript highlight mechanistic and structural insights for D. melanogaster AgmNAT, an enzyme that catalyzes the formation of N-acetylagmatine from acetyl-CoA and agmatine. We provide evidence for an underappreciated reaction in arginine metabolism; however, it still remains unclear if N-acetylation of agmatine by an N-acetyltransferase enzyme is biologically relevant. A combination of data provided herein and reported from other labs speaks to its relevancy, warranting further investigation into this chemical transformation as a part of arginine metabolism. Furthermore, we outline a chemical mechanism for the AgmNAT-catalyzed formation of N-acetylagmatine (and, by extension, other N-acylamides), which is consistent with the data presented herein. We also provide evidence for important active site residues involved in substrate binding and maintaining the structural integrity of the active site for efficient catalysis, though further work is necessary to provide more evidence for the dynamic nature of the AgmNAT active site.
Oligonucleotides were purchased from Eurofins MWG Operon. PfuUltra High-Fidelity DNA polymerase was purchased from Agilent. BL21 (DE3) E.coli cells and pET-28a(+) vector were purchased from Novagen. NdeI, XhoI, Antarctic Phosphatase, and T4 DNA ligase were purchased from New England Biolabs. Kanamycin monosulfate and IPTG were purchased from Gold Biotechnology. Acyl-CoAs were purchased from Sigma-Aldrich. Cayman Chemical commercially synthesized N 1 -acetylspermidine. All other reagents were of the highest quality and purchased from either Sigma-Aldrich or Fisher Scientific.
AgmNAT: sub-cloning, expression, and purification. AgmNAT was inserted into a pET-28a vector using NdeI and XhoI restriction sites, yielding the final expression vector: AgmNAT-pET-28a, that after transformation into E.coli BL21 (DE3) cells expressed a protein with an N-terminal His 6 -tag followed by a thrombin cleavage site. The E. coli BL21 (DE3) cells containing the AgmNAT-pET-28a vector was cultured using LB media supplemented with 40 μg/mL kanamycin at 37 °C. The culture was induced with 1.0 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at an OD 600 ~ 0.6, followed by an additional four hours at 37 °C. The final culture was harvested by centrifugation at 5,000 × g for 10 min at 4 °C and the pellet was collected. The pellet was resuspended in 20 mM Tris, pH 7.9, 500 mM NaCl, 5 mM imidazole, lysed by sonication, and then centrifuged at 10,000 × g for 15 min at 4 °C. The supernatant was collected and loaded onto 6 mL of ProBond ™ nickel-chelating resin, followed by two wash steps: wash one -10 column volumes of 20 mM Tris, pH 7.9, 500 mM NaCl, 5 mM imidazole followed by wash two -10 column volumes of 20 mM Tris, pH 7.9, 500 mM NaCl, 60 mM imidazole. AgmNAT was eluted in 1 mL fractions using 20 mM Tris, pH 7.9, 500 mM NaCl, 500 mM imidazole, the protein pooled, and extensively dialyzed at 4 °C against 20 mM Tris pH 7.4, 200 mM NaCl. The concentration of AgmNAT was determined using the Bradford assay indexed against BSA as a standard, and purity was assessed by a SDS-PAGE gel (proteins visualized using by Coomassie stain). Purification of recombinant AgmNAT by nickel affinity chromatography yielded pure protein (≥95%) as visualized by SDS-PAGE ( Supplementary Fig. S6).
AgmNAT crystallography. After nickel-affinity purification, 30 mg of AgmNAT was subjected to dialysis against 50 mM HEPES pH 8.2, 200 mM NaCl, followed by removal of the His 6 affinity-tag using 60 U of biotinylated thrombin for 18 h in a fresh batch of 50 mM HEPES pH 8.2, 200 mM NaCl leaving an unnatural Gly-Ser-His at the N-terminus. The protein mixture was again subjected to nickel-affinity chromatography to remove undigested AgmNAT. AgmNAT was eluted in the 20 mM Tris, pH 7.9, 500 mM NaCl, 60 mM imidazole fraction, whereas the His 6 -AgmNAT was retained on the column until eluted with 20 mM Tris, pH 7.9, 500 mM NaCl, 500 mM imidazole. The biotinylated thrombin was removed by using 3 mL of Pierce monomeric avidin agarose resin at 4 °C for 30 min, followed by centrifugation to recover AgmNAT, and AgmNAT concentrated to ~10 mg/mL by ultrafiltration. Further purification was performed using a HiTrap Q FF column with a linear gradient from 50 mM HEPES pH 8.2 to 50 mM HEPES pH 8.2, 0.5 M NaCl with AgmNAT eluting in fractions containing ~150 mM NaCl. A final SEC purification step was used after the ion exchange step and purified AgmNAT was concentrated to ~8 mg/ml in 50 mM HEPES pH 8.2, 100 mM NaCl for crystallization screening. The Phoenix crystallization robot and Qiagen screening kits were used to evaluate different crystallization conditions for AgmNAT. AgmNAT was crystallized using the hanging-drop vapor diffusion method in 100 mM Tris pH 8.0, 200 mM sodium acetate, 30% PEG 4000. The drop contained a 1:1 ratio of 1 μL of 8 mg/mL AgmNAT with 1 μL of well solution and incubated at 20 °C. Crystals were of elongated rod-shape. Diffraction was measured at the 22-ID-D SER-CAT beamline at the Advanced Photon Source (APS), Argonne, IL. Data were indexed, scaled, and merged with iMosflm using the CCP4 suite 80 . A homology model was constructed based on the AgmNAT sequence using the program SWISS-MODEL 81 with mosquito arylalkylamine N-acetyltransferase (PDB ID 4FD4) 21 as a template for molecular replacement. The molecular replacement program Phaser-MR was used in PHENIX. The models of refinement were first obtained using a rigid-body refinement using phenix.refine in PHENIX. PHENIX 82 and Coot 83 were used to complete the model rebuilding and refinement. For refinement, data was cut at 2.3 A due to relatively poor data quality at higher resolutions. The crystal structure has been deposited into the Protein Data Bank with accession code 5K9N.

Construction of AgmNAT site-directed mutants.
Site-directed mutants of AgmNAT were constructed by the overlap extension method. Using the primers shown in Table S1, each mutant was amplified using pfuUltra High-Fidelity DNA polymerase with the following PCR conditions: initial denaturing step of 95 °C for 2 min, then 30 cycles of 95 °C for 30 s; 60 °C annealing temperature for 30 s; 72 °C extension step for 1 min; then a final extension step of 72 °C for 10 min. Following the amplification of the AgmNAT site-directed mutant, the sub-cloning, expression, and purification procedures are the same as discussed for the wild-type enzyme.
Measurement of enzyme activity. Steady-state kinetic constants for AgmNAT were determined by measuring the rate of coenzyme A release using Ellman's reagent (DTNB) at 412 nm (molar absorptivity = 13,600 M −1 cm −1 ) [15][16][17] . The assay consisted of 300 mM Tris pH 8.5, 150 μM DTNB, and the desired concentration of acyl-CoA and amine substrates. Initial velocities were measured using a Cary 300 Bio UV-Visible spectrophotometer at 22 °C. Acyl-CoA kinetic constants were evaluated by holding the concentration of agmatine at a constant saturating concentration (5 mM). Amine kinetic constants were evaluated by holding the concentration of acetyl-CoA at a constant saturating concentration (500 μM). The apparent kinetic constants were determined by fitting the resulting data to equation 1 using SigmaPlot 12.0, where v o is the initial velocity, V max,app is the apparent maximal velocity, [S] is the substrate concentration, and K m,app is the apparent Michaelis constant. Each assay was Scientific RepoRts | 7: 13432 | DOI:10.1038/s41598-017-13669-6 performed in triplicate and the uncertainty for the k cat,app and (k cat /K m ) app values were calculated using equation 2, where σ is the standard error.
x 2 y 2 Kinetic mechanism and inhibitor analysis. Defining the kinetic mechanism of AgmNAT was accomplished by evaluating double reciprocal plots of initial velocity data for acetyl-CoA and agmatine, followed by determining the type of inhibition for substrate analogs used as dead-end inhibitors or N-acetylagmatine for product inhibition. Initial velocities were determined by varying the concentration of one substrate, while holding the other substrate at a fixed concentration. Acetyl-CoA was evaluated at 20, 50, 100, 250 and 500 μM, whereas agmatine was evaluated at 60, 300, 750 and 1500 μM. The resulting initial velocity data was fit to equation 3 for an ordered Bi-Bi mechanism and equation 4 for a ping pong mechanism using IGOR Pro 6. Inhibition experiments by either substrate analogs or N-acetylagamatine were used to discriminate between an ordered, random sequential, or ping pong kinetic mechanism. Oleoyl-CoA, arcaine, and L-arginine methyl ester were used as dead-end inhibitors for AgmNAT while N-acetylagmatine was used for product inhibition. Initial velocity patterns were generated by varying the concentration of one substrate, holding the other substrate concentration at its apparent K m , and changing the concentration of inhibitor for each data set in triplicate. The resulting data was fit to equations 5-7, for competitive, noncompetitive, and uncompetitive inhibition respectively using SigmaPlot 12.0. For equations 4-6, v o is the initial velocity, V max,app is the apparent maximal velocity, K m,app is the apparent Michaelis constant, [S] is the substrate concentration, [I] is the inhibitor concentration, and K i is the inhibition constant. Rate versus pH. The pH-dependence on the kinetic constants for acetyl-CoA was determined using intervals of 0.5 pH units, ranging from 6.5-9.5. Buffers used to measure the pH-dependence were MES (pH 6.5 and 7.0), Tris (pH 7.0-9.0), AmeP (pH 9.0 and 9.5). The resulting data were fit to equations 8 (log (k cat /K m ) app -acetyl-CoA and equation 9 (log k cat,app -acetyl-CoA ) to determine the apparent pK a values using IGOR Pro 6.34 A, where c is the pH-independent plateau. The wild-type enzyme is reported in triplicate, whereas the E34A mutant was evaluated in duplicate.
Agmatine. To a solution of putrescine (2.0 g, 22.7 mmol) in water (20 mL) was added 2-methylisouronium sulfate (2.7 g, 11 mmol). The mixture was heated to 50 °C for 6 hours, then cooled in an ice bath for 30 minutes. During this time, a white precipitate was formed, which was collected by filtration, and then washed with ice water to give agmatine (1.3 g, 44%) as a white solid that was used without further purification. 1 H NMR (500 MHz, D 2 O) δ 3.08 (t, J = 6.0 Hz, 2 H), 2.81 (t, J = 6.8 Hz, 2 H), 1.53 (br. s., 4 H) ppm. 13  N-Acetylagmatine. To a mixture of agmatine (1.0 g, 7.62 mmol) in pyridine (10 mL) was added acetyl chloride (542 μL, 7.62 mmol) dropwise. The mixture was allowed to stir at room temperature for 4 hours, then was concentrated on a rotary evaporator. The crude residue was adsorbed onto silica gel and purified by flash column chromatography (methylene chloride/methanol 19:1) to give N-acetylagmatine (400 mg, 30%) as a viscous, colorless oil. 1