Evolution of Insect Arylalkylamine N-Acetyltransferases: Structural Evidence from the Yellow Fever Mosquito, Aedes Aegypti

Home , Mosquito

Evolution of insect arylalkylamine N-acetyltransferases: Structural evidence from the yellow fever mosquito, Aedes aegypti

Qian Hana, Howard Robinsonb, Haizhen Dinga, Bruce M. Christensenc, and Jianyong Lia,1

aDepartment of Biochemistry, Virginia Tech, Blacksburg, VA 24061; bBiology Department, Brookhaven National Laboratory, Upton, NY 11973; and cDepartment of Pathobiological Sciences, University of Wisconsin, Madison, WI 53706

Edited by Alexander S. Raikhel, University of California, Riverside, CA, and approved June 6, 2012 (received for review April 23, 2012) Arylalkylamine N-acetyltransferase (aaNAT) catalyzes the transace- and darkened. The cuticle is critical for insect survival, and thus is tylation from acetyl-CoA to arylalkylamines. aaNATs are involved in one of the primary targets for insect pest and disease vector con- sclerotization and neurotransmitter inactivation in insects. Phyletic trol (13, 14). There are two sclerotization precursors, N-ace- distribution analysis confirms three clusters of aaNAT-like sequen- tyldopamine and N-β-alanyldopamine. Both are synthesized from ces in insects: typical insect aaNAT, polyamine NAT-like aaNAT, and dopamine, in a pathway starting with tyrosine and including ty- mosquito unique putative aaNAT (paaNAT). Here we studied three rosine hydroxylase and L-DOPA decarboxylase enzymes, among proteins: aaNAT2, aaNAT5b, and paaNAT7, each from a different others downstream (15, 16). N-acetyldopamine is one of the cluster. aaNAT2, a protein from the typical insect aaNAT cluster, aaNAT-catalyzed enzyme products. Therefore, aaNATs occupy uses histamine as a substrate as well as the previously identified a special position in insect cuticle sclerotization. Biochemical and arylalkylamines. aaNAT5b, a protein from polyamine NAT -like structural characterization of insect aaNATs may provide insight aaNAT cluster, uses hydrazine and histamine as substrates. The into the in vivo functions of these enzymes and thus a means to crystal structure of aaNAT2 was determined using single-wave- manipulate enzyme activities to control the sclerotization process. length anomalous dispersion methods, and that of native aaNAT2, Up to now, no single aaNAT crystal structure from any insect has aaNAT5b and paaNAT7 was detected using molecular replacement ever been solved. Although the crystal structure of an aaNAT, also techniques. All three aaNAT structures have a common fold core known as serotonin N-acetyltransferase (SNAT), from Ovis aries of GCN5-related N-acetyltransferase superfamily proteins, along β β was solved previously (17), it shares only 2% and 7% sequence with a unique structural feature: helix/helices between 3 and 4 identity with A. aegypti aaNAT2 and aaNAT1, respectively (2). strands. Our data provide a start toward a more comprehensive Although 13 putative aaNATs have been identified by a BLAST understanding of the structure–function relationship and physiol- search (2), most of them have not been biochemically identified as ogy of aaNATs from the mosquito Aedes aegypti and serve as a ref- aaNAT. In this report, we provide crystal structures for aaNAT2, erence for studying the aaNAT family of proteins from other insect aaNAT5b, and putative aaNAT7 (paaNAT7) and screen more species. The structures of three different types of aaNATs may pro- possible substrates for these three enzymes. vide targets for designing insecticides for use in mosquito control. Results and Discussion biogenic amine | melatonin synthesis | catecholamine | insect cuticle Evolution and Substrate Screening of Insect Putative aaNATs. Mam- mal, avian, and anuran genomes possess a single copy of the aaNAT rylalkylamine N-acetyltransferases (aaNATs) catalyze the gene (18). Teleost fish have up to three paralogs (18), and amphioxus Atransacetylation of acetyl-CoA to arylalkylamines (1, 2). In has seven aaNAT homolog genes (19). Insects do not have mam- mammals, aaNAT is involved primarily in the synthesis of N- malian aaNAT homologs, based on sequence similarity searches of BIOCHEMISTRY acetylserotonin. The N-acetylation of serotonin (5-hydroxytrypta- sequenced genomes and phylogenetic analysis (18, 19); however, mine) is a rate-limiting step for the synthesis of melatonin in the insects do have multiple aaNAT enzymes that show very low se- pineal gland. Physiologically, aaNAT regulates circadian rhythm in quence identity with mammalian aaNATs (2, 7, 12). The identified mammals (3, 4). In insects, aaNATs play multiple roles. In addition insect aaNAT sequences and the available genome sequences for to melatonin synthesis, aaNAT enzymes are involved in aromatic a number of insect species make it possible to predict hypothetical neurotransmitter inactivation and cuticle sclerotization (5). aaNATs aaNAT sequences in insects via a bioinformatics approach. inactivate arylalkylamines, such as octopamine, dopamine, and se- A BLAST search using the activity-verified D. melanogaster rotonin (6, 7), because insects lack the monoamine oxidase (8) used aaNAT sequence against three currently available mosquito spe- by mammals to inactivate arylalkylamines (9). Studies dealing with cies (A. aegypti, Anopheles gambiae,andCulex quinquefasciatus) an aaNAT from Bombyx mori clearly established its function in adult and three nonmosquito insect species, identified 8 sequences from cuticle sclerotization through the production of N-acetyldopamine, A. gambiae, 13 from A. aegypti,15fromC. quinquefasciatus,13 a key cuticle protein crosslinking precursor (10, 11). from D. melanogaster, 3 from B. mori, and 1 from Periplaneta Unlike mammals, insects have evolved multiple aaNATs (2, 7, 12). In the yellow fever mosquito, Aedes aegypti, 13 putative aaNATs have been identified by a BLAST search using a positively verified Dro- Author contributions: Q.H. and J.L. designed research; Q.H., H.R., H.D., and J.L. performed sophila melanogaster aaNAT sequence; two of these aaNATs have research; Q.H., H.R., and J.L. contributed new reagents/analytic tools; Q.H., H.R., B.M.C., been biochemically identified as aaNAT1 and aaNAT2 (2). The and J.L. analyzed data; and Q.H., B.M.C., and J.L. wrote the paper. presence of multiple aaNATs in mosquitoes suggests the importance The authors declare no conflict of interest. of arylalkylamine acetylation in these insect species. This article is a PNAS Direct Submission. The cuticular exoskeleton provides insects with protection Database deposition: Crystallography, atomic coordinates, and structure factors have against physical injury and infection, rigidity for muscle attach- been deposited in the Protein Data Bank, http://www.rcsb.org/pdb/home/home.do (acces- ment, and mechanical support and flexibility for joint movement. sion nos. 4fd4, 4fd5, 4fd6 and 4fd7). During development and growth, insects periodically shed their 1To whom correspondence should be addressed. E-mail: [email protected]. old cuticle and produce a new one. New cuticle formation involves This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. sclerotization, a process in which the cuticle becomes hardened 1073/pnas.1206828109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1206828109 PNAS | July 17, 2012 | vol. 109 | no. 29 | 11669–11674 Downloaded by guest on September 23, 2021 americana showing recognizable similarity. To understand the activity of aaNAT2 and aaNAT5b clearly demonstrate that phylogenetic relationship among the putative aaNATs in insects, A. aegypti has histamine NAT. we constructed a phylogenetic tree (Fig. 1). Phyletic distribution We also screened 61 possible substrates for paaNAT7, one of analysis confirmed three major clusters in insects, termed clusters the proteins in cluster 3, but detected no transacetylation activity 1, 2, and 3. The proteins in cluster 1 likely represent typical insect (Table S1). It must be noted that cluster 3 proteins might be aaNATs, because all positively identified insect aaNATs are lo- unique to mosquitoes, however, given that the other insects cated in this cluster. aaNAT1 and aaNAT2 from A. aegypti are studied have no putative aaNAT sequences in the cluster. classified in this cluster. By screening more substrates, we identified two more substrates of aaNAT2, histamine, and bromoe- Overall Structures of aaNAT2, aaNAT5b, and paaNAT7. An aaNAT2i thylamine (Tables S1 and S2). No proteins have yet been identified (i.e., aaNAT2 crystal soaking with iodine ions) crystal was diffracted as aaNATs in clusters 2 and 3. We found that one of the proteins in to 1.64 Å. Seven theoretical iodine sites were found in aaNAT2i cluster 2, aaNAT5b, uses histamine, hydrazine, and hexamethy- with hkl2map, giving an overall figure of merit of 0.464 as calculated lenediamine as substrates (Tables S1 and S2). Thus, cluster 2 by PHENIX.Autosol. A further PHENIX run using AutoBuild re- proteins represent a different type of aaNATs found in insects, and sulted in an initial model with approximately 80% of the residues we have named these polyamine NAT-like aaNATs based on their built. This model was subjected to iterative cycles of refinement substrate profiles (20). Although both aaNAT2 and aaNAT5b use and model rebuilding until the final model reached acceptable histamine as a substrate, their pH profiles are quite different (Fig. parameters in geometry and fitting. aaNAT2n (i.e., native crystal of S1). The finding of histamine NAT activity is intriguing. In aaNAT2), aaNAT5b, and paaNAT7 crystal structures were solved arthropods, histamine is an important neurotransmitter, particu- by molecular replacement, using the solved aaNAT2i structure as a larly in photoreceptors. In Drosophila visual tissues, histamine is search model. The data collection, refinement statistics, and sta- metabolized to carcinine (β-alanyl histamine) by Ebony (21, 22). In tistics on Ramachandran plots as definedwithPROCHECK(26) visual tissues of the horseshoe crab Limulus polyphemus,most of four crystal structures are provided in Table S3. histamine is metabolized to imidazole acetic acid and γ-glutamyl The overall structures of aaNAT2, aaNAT5b, and aaNAT7 histamine (23). Although N-acetylation of histamine in arthropods appear similar even though they have low sequence identity among has not been studied, N-acetyl histamine has been detected in one another (Table S4). Overall, the structures of aaNAT2 and locusts and Drosophila (24, 25), suggesting that arthropods might aaNAT5b contain seven β-strands (β1–β7), and the structure of have a histamine NAT. The identification of histamine acetylation paaNAT7 contains eight β-strands (β0–β7). In all three proteins,

Fig. 1. A phylogenetic tree of identiﬁed and putative aaNATs from mosquitoes and other insects. Sequences were aligned using ClustalW, and the dendrogram was generated using the neighbor-joining method. Numbers at nodes show bootstrap values based on 1,000 replicates. The National Center for Biotechnology Information gi number is provided for each protein sequence. The three proteins for which crystal structures were solved in this report are shown with a gray background. Aea, Aedes aegypti; Ag, Anopheles gambiae; Bm, Bombyx mori; Cq, Culex quinquefasciatus; Dm, Drosophila melanogaster;Pa, Periplaneta americana.

11670 | www.pnas.org/cgi/doi/10.1073/pnas.1206828109 Han et al. Downloaded by guest on September 23, 2021 the β-strands are mostly antiparallel to one another and form a search with the aaNAT5b structure found that aaNAT5b is most semicylindrical β-sheet. The C-terminal ends of the neighboring similar to histone acetyltransferase HPA2 of C. glutamicum strands β4andβ5 (the only two parallel β-strands in the sheet) are (PDB code 2qec; Z-score = 15.3; rmsd = 2.8) (DOI:10.2210/ splayed apart from each other (Fig. 2), which splits the β-sheet into pdb2qec/pdb,) PaiA from Bacillus subtilis (PDB code 1tiq; Z- two groups of antiparallel β-strands composed of β0/β1–β4and score = 15.2; rmsd = 3.3) (32), and a phosphinothricin acetyl- β5–β7. The tunnel thus created in the center of the β-sheet is transferase from Agrobacterium tumefaciens (PDB code 1yr0; necessary for the binding of the CoA cofactor and the substrate in Z-score = 15.40; rmsd = 2.8) (DOI:10.2210/pdb1yr0/pdb). other GCN5-related NAT (GNAT) proteins (27). All three pro- Comparison of aaNAT5b and the SNAT from O. aries showed teins have four conserved sequence motifs, termed C, D, A, and B a Z-score = 13.5 and rmsd = 2.9. A Dali search with paaNAT7 in N- to C-terminal order, and contain a common fold core that structure revealed that paaNAT7 is most similar to histone includes α-helices on both sides of six-stranded mixed β-sheets acetyltransferase HPA2 of C. glutamicum (PDB code 2qec; Z- (β1–β6) of the GNAT superfamily (28) (Fig. 2). Similar to the score = 14.3; rmsd = 2.8) (DOI:10.2210/pdb2qec/pdb,) the SNAT structure, all three mosquito aaNATs have an additional β acetyltransferase GNAT family (NP_688560.1) from Streptococ- strand, β7, at the C-terminus. In addition, paaNAT7 has one more cus agalactiae 2603 (PDB code 2pc1; Z-score = 14.2; rmsd = 3.1) extra β strand, β0, at the N-terminus. (DOI:10.2210/pdb2pc1/pdb,) and a putative GNAT Ta0374 from Thermoplasma acidophilum (PDB code 3ﬁx; Z-score = 14.1; Structural Comparison of all Three Structures and Other Selected rmsd = 2.5) (33). Comparison of paaNAT7 and the SNAT from GNAT Superfamily Structures. A Dali search (29) with the aaNAT2 O. aries revealed a Z-score = 12.0 and rmsd = 3.2. structure revealed that aaNAT2 is most similar to histone ace- Although GNAT superfamily members have four conserved tyltransferase HPA2 of Corynebacterium glutamicum (PDB code sequence motifs (34) and contain a common fold core (28), they all 2qec; Z-score = 15.4; rmsd = 3.1) (DOI:10.2210/pdb2qec/pdb,) display limited sequence similarity and retain few invariant residues N-terminal acetylase ARD1 from Sulfolobus solfataricus P2 (27), particularly in the regions where a more inconsistent stock of (PDB code 2x7b; Z-score = 15.4; rmsd = 2.6) (30), and ribo- structures is built around the common core. This makes homology somal S18 N-α-protein acetyltransferase from Salmonella typhi- detection a challenge for GNAT superfamily members using se- murium LT2 (PDB code 2cnt; Z-score = 15.4; rmsd = 2.2) (31). quence comparison alone (27, 35). Because the characteristic Comparison of aaNAT2 and the SNAT from O. aries (PDB code GNAT fold is well conserved (36), we used the 3D structural 1kuy) (17) revealed a Z-score = 13.4 and rmsd = 2.9. A Dali alignment to compare three mosquito aaNATs and other related

Fig. 2. Overall structures of aaNAT2, BIOCHEMISTRY aaNAT5b and paaNAT7 and 3D structural alignment of above three proteins and SNAT from O. aries.(A) Cartoon representation of aaNAT2. Motifs C, D, A, and B are labeled in purple, green, yellow, and red, respectively. Other regions of the structure are in gray. β-strands are numbered β1–β7. (B) Cartoon representation of aaNAT5b. The same scheme as used in A is used to color and label motifs, other parts of the structure, and β strands. (C) Cartoon representation of paaNAT7. The same scheme as used in A is used to color and label motifs and other parts of the structure. β strands are labeled β0–β7. (D) Superposition of the three mosquito aaNAT structures onto the structure of SNAT from O. aries. The same scheme as used in A is used to color and label motifs and β strands. Regions with major structural differences are colored gray for mosquito aaNATs and cyan for SNAT. (E) 3D structural alignment of aaNAT2, aaNAT5b, paaNAT7, and SNAT from O. aries (Oa-aaNAT). The second structures of SNAT are labeled under the sequences, and motifs C, D, A, and B are in purple, green, yellow, and red background boxes.

Han et al. PNAS | July 17, 2012 | vol. 109 | no. 29 | 11671 Downloaded by guest on September 23, 2021 fold cores are nearly universally conserved (Fig. 3A)despite structure-based sequence alignments showing low pairwise sequence identity. Phyletic distribution analysis identiﬁed three mosquito aaNATs located in close proximity in the phylogenetic tree based on the 3D structural alignment and different from gpNAT, AAC, and mammalian aaNAT, SNAT. Interestingly, mosquito aaNATs are structurally close to HAT (Fig. 3B). Careful examination of the structural features of all three mosquito aaNATs revealed a unique structural feature, the helix/helices between β3andβ4 (Fig. 4). aaNAT2 and aaNAT5b have two helices, and paaNAT7 has one helix. Future investigations into the possible role of this structural feature might prove quite interesting.

Catalytic Funnels and Cofactor Binding. GNAT superfamily proteins catalyze the transfer of an acetyl group to their substrates. The kinetic mechanism of most GNATs involves the ordered formation of a ternary complex. The reaction begins with acetyl-CoA binding, followed by binding of substrate, then direct transfer of the acetyl group from acetyl-CoA to the substrate, and finally product and subsequent CoA release (27). His120, His122, Leu124, and Tyr168 play important roles in the enzyme catalysis of sheep SNAT (36, 50), and by superimposing the three mosquito (putative) aaNATs onto the SNAT structure, we identified the equivalent residues in mosquito aaNATs. aaNAT5b shares three of the four residues, whereas aaNAT2 and paaNAT7 share only one of the four residues (Fig. 5). It has been suggested that Tyr168 in sheep SNAT functions as a general acid, serving to protonate the thiolate anion of CoA after decomposition of the tetrahedral intermediate formed by attack of the primary amine of serotonin on the acetyl-CoA thioester (36). However, aaNAT2, aaNAT5b, and paaNAT7 have Val180, Ala176, and Ala197, respectively, at the Tyr168 position in SNAT, suggesting that mosquito aaNATs likely use a different mechanism in catalysis. We evaluated general surface and electrostatic properties to as- sess the similarities and differences in the catalytic funnels of the Fig. 3. Superposition of aaNAT2, aaNAT5b, aaNAT7, and other represen- four enzymes, aaNAT2, aaNAT5b, paaNAT7, and SNAT (Fig. tative GNAT structures and the phylogenetic tree. (A) Superposition of eight S2). Interestingly, aaNAT2 had a wider catalytic funnel than GNAT structures (PDB codes 1PUA, 1I21, 1S3Z, 1CJW, and 1M4G, as well as aaNAT5b, which might explain why aaNAT5b catalyzes only the three mosquito putative aaNATs) reveals that GNAT common fold core smaller substrates, hydrazine and histamine. The surface of the structures are nearly universally conserved despite structure-based sequence paaNAT7 catalytic funnel was more positively charged, suggest- alignments that reveal very low pairwise sequence identity. Motifs C, D, A, ing that paaNAT7 may use a more negatively charged substrate. and B are well superposed, labeled as C, D, A, and B, respectively. (B) The This possibility should be considered in future substrate screening phylogenetic tree based on the 3D structural alignment of 15 GNAT struc- fi tures, including aaNAT2, aaNAT5b, and paaNAT7. After 3D alignment, the for aaNAT7. Using xed molecular docking, we obtained binding “PhylogeneticTree_TreeTop” function in STRAP was used to draw a cluster positions of CoA molecules in aaNAT2, aaNAT5b, and paaNAT7 algorithm-type phylogenetic tree. (Fig. S3). paaNAT7 was seen to have a similar CoA-binding

GNAT superfamily proteins. GNATs catalyze a diverse arrays of substrates, including N-acetylation of lysine residues of proteins (histones and transcription factors) and small molecules such as polyamines, aminoglycosides, and arylalkylamines (37). Based on substrate specificities, a number of major GNAT superfamily proteins have been identified, including histone NAT (HAT), aminoglycoside NAT (AAC), aaNAT, and glucosamine 6-phosphate NAT (gpNAT). We chose representative structures from the foregoing enzymes for a structural comparison: a HAT from yeast (MYST; PDB code 1fy7) (38), a HAT from Tetrahymena (Gcn5/ PCAF; PDB code 1puA) (39), human HAT (p300/CBP; PDB code 3biy) (40), a different HAT from yeast (Rtt109; PDB code 3qm0) (41), an AAC6 from Enterococcus faecium (PDB code 1n71) (42), an AAC6 from Salmonella enterica (PDB code 1s3z) (43), an AAC2 from Mycobacterium tuberculosis (PDB code 1m4g) (44), a gpNAT Fig. 4. Unique structural features of aaNAT2, aaNAT5b, and paaNAT7. (A– β β from Trypanosoma (PDB code 3i3g) (45), human gpNAT (PDB C) Unique structural features between 3 and 4 strands of aaNAT2 (A), code 3cxs) (46), a gpNAT from Aspergillus fumigatus (PDB code aaNAT5b (B), and paaNAT7 (C). (D) Superposition of 15 GNAT structures, including aaNAT2, aaNAT5b, and paaNAT7. Only the three mosquito aaNATs 2vxk) (47), a gpNAT from yeast (PDB code 1i21) (48), and SNAT have this unique helix/helices structural feature; the other 12 GNATs, rep- from O. aries (PDB code 1cjw) (49). Superposition of five GNAT resenting different HATs, different AACs, different GNATs, and SNAT shown structures and three mosquito aaNATs revealed that the common in Fig. 3, do not have this structural feature.

11672 | www.pnas.org/cgi/doi/10.1073/pnas.1206828109 Han et al. Downloaded by guest on September 23, 2021 added, AlaGlyHis in the N-termini, because of the extra nucleotide sequence in the Impact-CN plasmid. The recombinant proteins were then produced in Escherichia coli cells and puriﬁed as described previously (2). The purity of the recombinant proteins was assessed by the presence of a single band with anticipated molecular weight on an SDS/PAGE gel. Protein concentrations were determined using a Bio-Rad protein assay kit with BSA as a standard.

Phylogenetic Analysis of aaNATs and Putative aaNATs in Insect Species. Insect aaNAT homologs from different insect species were identiﬁed through a search of the nonredundant database of protein sequences using the BLAST and PSI-BLAST search features (52). Putative aaNAT sequences from A. aegypti, A. gambiae, C. quinquefasciatus, D. melanogaster, B. mori,and P. americana were selected, and a multiple sequence alignment of the re- spective protein sequences was constructed using the MEGA4-ClustalW alignment program (53). A maximum likelihood phylogenetic tree was constructed using the neighbor-joining method and bootstrap analysis (53).

Fig. 5. Catalytic residues of aaNAT2, aaNAT5b, paaNAT7, and SNAT (PDB Crystallization. The crystals were grown by hanging-drop vapor diffusion code 1cjw). Parts of motifs A and B are shown in ribbons, colored cyan for methods with a 150-μL volume of reservoir solution at and a 2-μL drop aaNAT2, yellow for aaNAT5b, blue for paaNAT7, and green for SNAT. The volume, containing 1 μL of protein sample and 1 μL of reservoir solution. The residues involved in the enzyme catalysis in SNAT and the equivalent resi- optimized crystallization buffer for aaNAT2 consisted of 0.17 M ammonium dues in other three mosquito (putative) aaNATs are shown as sticks, with red acetate, 25.5% polyethylene glycol 4000, and 17% glycerol, and 0.1 M so- oxygen and blue nitrogen atoms. Carbon atoms and residue labels are dium citrate (pH 5.6). For aaNAT5b, the buffer consisted of 0.5 M sodium shown using the same color scheme as for the ribbon presentations. chloride, 25.5% polyethylene glycol 8000, 15% glycerol, and 0.1 M sodium acetate (pH 4.6). The buffer for paaNAT7 comprised 2 M ammonium sulfate, funnel as aaNAT2 and aaNAT5b, demonstrating its character- 5% polyethylene glycol 400, and 0.1 M Hepes sodium (pH 7.5). aaNAT2 and fi aaNAT5b crystals were mounted without the addition of extra cryoprotec- istics as an NAT, although we have not yet identi ed its substrate. tant agents. The iodine derivative of paaNAT7 was cryogenized for 2 min fi using 22% ethylene glycol in the crystallization buffer as a cryoprotectant Potential Biological Implications. Mosquitoes transmit malaria, - solution, which also contained 0.5 M sodium iodine. The iodine derivative of larial worms, and numerous arboviruses (e.g., dengue fever, West aaNAT2 (aaNAT2i) was prepared by soaking a single crystal in the crystalli- Nile virus), which are major threats to human health throughout zation buffer, which contained 0.5 M sodium iodine, for 2 min. much of the world. Among these, malaria is considered the most prevalent life-threatening disease, with annual estimates of new Data Collection and Processing. Crystal diffraction data were collected at the cases ranging from 300 million to 660 million (51). aaNATs play Brookhaven National Synchrotron Light Source beam line X29A (λ =1.075 or critical roles in mosquito survival and development, including bio- 1.540 Å). Data were collected using an ADSC Q315 CCD detector. All data genic amine and polyamine detoxification, melatonin synthesis, and were indexed and integrated using HKL-2000 software. Scaling and merging cuticle formation. The identification of mosquito aaNATs provides of diffraction data were performed using the SCALEPACK program (54). The a starting point for more comprehensive investigations of the bio- parameters of the crystals and data collection are listed in Table S3. chemistry and physiology of aaNATs in mosquitoes and serves as Structure Determination. The structure of aaNAT2 was determined by the SAD a reference for studying the aaNAT family of proteins in other phasing technique withiodine anomaloussignals.Heavy atom siteswere located insect species. The crystal structures of the three different types of using hkl2map (55), and the initial SAD phases were calculated by PHENIX. mosquito aaNATs that we have identified should aid the design of Autosol (56). Initial models were built using PHENIX.AutoBuild (56). The model new types of insecticide compounds aimed at affecting mosquito was refined using SAD refinement with optimization of the iodide ion occu- aaNAT activity or stability. Mosquito aaNATs differ greatly from pancy in Refmac 5.2 (57). Final models were produced after numerous iterative mammalian aaNATs in both evolution and molecular structure, rounds of manual rebuilding in Coot (58) and refinement in Refmac 5.2 (57). have a unique structural feature (the helix/helices between β3and The structures of native aaNAT2, aaNAT5b (21% sequence identical with BIOCHEMISTRY β4 strands) and may have a different catalytic mechanism. Thus, aaNAT2), and paaNAT7 (19% sequence identical with aaNAT2) were de- inhibitors of mosquito aaNATs might not affect mammalian termined by the molecular replacement method using the solved aaNAT2 aaNATs. Potent mosquito aaNAT inhibitors might provide another structure. The Molrep (59) and Phaser (60) programs were used to calculate both the cross-rotation and translation of the model. The initial model was pathway to effective mosquito control. However, more work is fi fi subjected to iterative cycles of crystallographic re nement with Refmac 5.2 (57) needed to de ne the catalytic mechanisms of aaNAT2, aaNAT5b, and graphic sessions for model building using Coot (58). Solvent molecules were and paaNAT7 and identify substrates of paaNAT7. automatically added and refinedwithARP/warp(61)togetherwithRefmac5.2.

Materials and Methods Structure Analysis. Superposition of structures was done using Lsqkab (62) in Expression and Purification of A. aegypti, aaNAT-2, aaNAT-5b, and paaNAT-7. the CCP4 suite. Figures were generated using Pymol (63). Protein surface Amplification of full-length cDNA for three proteins—aaNAT2 (GenBank ac- properties were analyzed using Pymol (63). 3D structural alignment and cession no. XP_001663122), aaNAT5b (GenBank accession no. XP_001649916), creation of the phylogenetic tree based on this 3D alignment were done and paaNAT7 (GenBank accession no. XP_001663019)—was achieved using using STRAP (64). the forward and reverse primers corresponding to 5′ and 3′ regions of the fi coding sequences (2). Their ampli ed cDNA sequences were cloned into an ACKNOWLEDGMENTS. This work was supported by National Institutes of Impact-CN plasmid (New England Biolabs) for expression of recombinant Health Grant AI 19769 and carried out in part at the National Synchrotron proteins. All recombinant proteins had three extra amino acid residues Light Source, Brookhaven National Laboratory.

1. Evans DA (1989) N-acetyltransferase. Pharmacol Ther 42:157–234. 5. Smith TJ (1990) Phylogenetic distribution and function of arylalkylamine N-acetyl- 2. Mehere P, Han Q, Christensen BM, Li J (2011) Identiﬁcation and characterization of transferase. Bioessays 12:30–33. two arylalkylamine N-acetyltransferases in the yellow fever mosquito, Aedes aegypti. 6. Brodbeck D, et al. (1998) Molecular and biochemical characterization of the aaNAT1 Insect Biochem Mol Biol 41:707–714. (Dat) locus in Drosophila melanogaster: Differential expression of two gene products. 3. Klein DC (2006) Evolution of the vertebrate pineal gland: The AANAT hypothesis. DNA Cell Biol 17:621–633. Chronobiol Int 23:5–20. 7. Amherd R, Hintermann E, Walz D, Affolter M, Meyer UA (2000) Puriﬁcation, cloning, 4. Klein DC (2007) Arylalkylamine N-acetyltransferase: “The timezyme.” J Biol Chem 282: and characterization of a second arylalkylamine N-acetyltransferase from Drosophila 4233–4237. melanogaster. DNA Cell Biol 19:697–705.

Han et al. PNAS | July 17, 2012 | vol. 109 | no. 29 | 11673 Downloaded by guest on September 23, 2021 8. Bortolato M, Chen K, Shih JC (2008) Monoamine oxidase inactivation: From patho- 37. Abo-Dalo B, Ndjonka D, Pinnen F, Liebau E, Lüersen K (2004) A novel member of the physiology to therapeutics. Adv Drug Deliv Rev 60:1527–1533. GCN5-related N-acetyltransferase superfamily from Caenorhabditis elegans prefer- 9. Tsugehara T, Iwai S, Fujiwara Y, Mita K, Takeda M (2007) Cloning and characterization entially catalyses the N-acetylation of thialysine [S-(2-aminoethyl)-L-cysteine]. Bio- of insect arylalkylamine N-acetyltransferase from Bombyx mori. Comp Biochem chem J 384:129–137. Physiol B Biochem Mol Biol 147:358–366. 38. Yan Y, Barlev NA, Haley RH, Berger SL, Marmorstein R (2000) Crystal structure of yeast 10. Dai FY, et al. (2010) Mutations of an arylalkylamine-N-acetyltransferase, Bm-iAANAT, Esa1 suggests a unified mechanism for catalysis and substrate binding by histone are responsible for silkworm melanism mutant. J Biol Chem 285:19553–19560. acetyltransferases. Mol Cell 6:1195–1205. 11. Zhan S, et al. (2010) Disruption of an N-acetyltransferase gene in the silkworm reveals 39. Clements A, et al. (2003) Structural basis for histone and phosphohistone binding by a novel role in pigmentation. Development 137:4083–4090. the GCN5 histone acetyltransferase. Mol Cell 12:461–473. 12. Hintermann E, Grieder NC, Amherd R, Brodbeck D, Meyer UA (1996) Cloning of an 40. Liu X, et al. (2008) The structural basis of protein acetylation by the p300/CBP tran- arylalkylamine N-acetyltransferase (aaNAT1) from Drosophila melanogaster ex- scriptional coactivator. Nature 451:846–850. pressed in the nervous system and the gut. Proc Natl Acad Sci USA 93:12315–12320. 41. Tang Y, et al. (2008) Fungal Rtt109 histone acetyltransferase is an unexpected 13. Sheng J, et al. (2006) Cloning a cuticle-degrading serine protease gene with biologic structural homolog of metazoan p300/CBP. Nat Struct Mol Biol 15:998. control function from Beauveria brongniartii and its expression in Escherichia coli. 42. Burk DL, Ghuman N, Wybenga-Groot LE, Berghuis AM (2003) X-ray structure of the – Curr Microbiol 53:124 128. AAC(6′)-Ii antibiotic resistance enzyme at 1.8 A resolution: Examination of oligomeric 14. Luz C, Batagin I (2005) Potential of oil-based formulations of Beauveria bassiana to arrangements in GNAT superfamily members. Protein Sci 12:426–437. – control Triatoma infestans. Mycopathologia 160:51 62. 43. Vetting MW, Magnet S, Nieves E, Roderick SL, Blanchard JS (2004) A bacterial ace- 15. Andersen SO (2010) Insect cuticular sclerotization: A review. Insect Biochem Mol Biol tyltransferase capable of regioselective N-acetylation of antibiotics and histones. – 40:166 178. Chem Biol 11:565–573. 16. Han Q, Ding H, Robinson H, Christensen BM, Li J (2010) Crystal structure and substrate 44. Vetting MW, Hegde SS, Javid-Majd F, Blanchard JS, Roderick SL (2002) Aminoglyco- fi speci city of Drosophila 3,4-dihydroxyphenylalanine decarboxylase. PLoS ONE 5: side 2′-N-acetyltransferase from Mycobacterium tuberculosis in complex with co- e8826. enzyme A and aminoglycoside substrates. Nat Struct Biol 9:653–658. 17. Wolf E, De Angelis J, Khalil EM, Cole PA, Burley SK (2002) X-ray crystallographic 45. Mariño K, et al. (2011) Characterization, localization, essentiality, and high-resolution studies of serotonin N-acetyltransferase catalysis and inhibition. J Mol Biol 317: crystal structure of glucosamine 6-phosphate N-acetyltransferase from Trypanosoma 215–224. brucei. Eukaryot Cell 10:985–997. 18. Coon SL, Klein DC (2006) Evolution of arylalkylamine N-acetyltransferase: Emergence 46. Wang J, Liu X, Liang YH, Li LF, Su XD (2008) Acceptor substrate binding revealed by and divergence. Mol Cell Endocrinol 252:2–10. crystal structure of human glucosamine-6-phosphate N-acetyltransferase 1. FEBS Lett 19. Pavlicek J, et al. (2010) Evolution of AANAT: Expansion of the gene family in the 582:2973–2978. cephalochordate amphioxus. BMC Evol Biol 10:154. 47. Hurtado-Guerrero R, et al. (2008) Structural and kinetic differences between human 20. Aisien SO, Walter RD (1993) Biogenic-amine acetylation: An additional function of the and Aspergillus fumigatus D-glucosamine-6-phosphate N-acetyltransferase. Biochem N-acetyltransferase from Fasciola hepatica. Biochem J 291:733–737. J 415:217–223. 21. True JR, et al. (2005) Drosophila tan encodes a novel hydrolase required in pigmen- 48. Peneff C, Mengin-Lecreulx D, Bourne Y (2001) The crystal structures of Apo and tation and vision. PLoS Genet 1:e63. complexed Saccharomyces cerevisiae GNA1 shed light on the catalytic mechanism of 22. Richardt A, et al. (2003) Ebony, a novel nonribosomal peptide synthetase for beta- an amino-sugar N-acetyltransferase. J Biol Chem 276:16328–16334. alanine conjugation with biogenic amines in Drosophila. J Biol Chem 278: 49. Hickman AB, Namboodiri MA, Klein DC, Dyda F (1999) The structural basis of ordered 41160–41166. substrate binding by serotonin N-acetyltransferase: Enzyme complex at 1.8 A reso- 23. Battelle BA, Hart MK (2002) Histamine metabolism in the visual system of the lution with a bisubstrate analog. Cell 97:361–369. horseshoe crab Limulus polyphemus. Comp Biochem Physiol A Mol Integr Physiol 133: 50. Scheibner KA, De Angelis J, Burley SK, Cole PA (2002) Investigation of the roles of 135–142. catalytic residues in serotonin N-acetyltransferase. J Biol Chem 277:18118–18126. 24. Elias MS, Evans PD (1983) Histamine in the insect nervous system: Distribution, synthesis and metabolism. J Neurochem 41:562–568. 51. Snow RW, Guerra CA, Noor AM, Myint HY, Hay SI (2005) The global distribution of – 25. Sarthy PV (1991) Histamine: A neurotransmitter candidate for Drosophila photo- clinical episodes of Plasmodium falciparum malaria. Nature 434:214 217. receptors. J Neurochem 57:1757–1768. 52. Altschul SF, et al. (1997) Gapped BLAST and PSI-BLAST: A new generation of protein – 26. Laskowski RA, Macarthur MW, Moss DS, Thornton JM (1993) Procheck: A program to database search programs. Nucleic Acids Res 25:3389 3402. check the stereochemical quality of protein structures. J Appl Cryst 26:283–291. 53. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics – 27. Dyda F, Klein DC, Hickman AB (2000) GCN5-related N-acetyltransferases: A structural Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596 1599. overview. Annu Rev Biophys Biomol Struct 29:81–103. 54. Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in os- – 28. Wybenga-Groot LE, Draker K, Wright GD, Berghuis AM (1999) Crystal structure of an cillation mode. Methods Enzymol 276:307 326. aminoglycoside 6′-N-acetyltransferase: Defining the GCN5-related N-acetyltransfer- 55. Pape T, Schneider TR (2004) HKL2MAP: A graphical user interface for phasing with – ase superfamily fold. Structure 7:497–507. SHELX programs. J Appl Cryst 37:843 844. 29. Holm L, Kaariainen S, Wilton C, Plewczynski D (2006) Using Dali for structural com- 56. Adams PD, et al. (2010) PHENIX: A comprehensive Python-based system for macro- – parison of proteins. Curr Protoc Bioinform Chap 5:Unit 5.5. molecular structure solution. Acta Crystallogr D Biol Crystallogr 66:213 221. fi 30. Oke M, et al. (2010) The Scottish Structural Proteomics Facility: Targets, methods and 57. Murshudov GN, Vagin AA, Dodson EJ (1997) Re nement of macromolecular struc- outputs. J Struct Funct Genomics 11:167–180. tures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53: 31. Vetting MW, Bareich DC, Yu M, Blanchard JS (2008) Crystal structure of RimI from 240–255. Salmonella typhimurium LT2, the GNAT responsible for N(alpha)-acetylation of ri- 58. Emsley P, Cowtan K (2004) Coot: Model-building tools for molecular graphics. Acta bosomal protein S18. Protein Sci 17:1781–1790. Crystallogr D Biol Crystallogr 60:2126–2132. 32. Forouhar F, et al. (2005) Structural and functional evidence for Bacillus subtilis PaiA as 59. Vagin A, Teplyakov A (1997) MOLREP: An automated program for molecular re- a novel N1-spermidine/spermine acetyltransferase. J Biol Chem 280:40328–40336. placement. J Appl Cryst 30:1022–1025. 33. Filippova EV, et al. (2011) Crystal structure of the novel PaiA N-acetyltransferase from 60. McCoy AJ, et al. (2007) Phaser crystallographic software. J Appl Cryst 40:658–674. Thermoplasma acidophilum involved in the negative control of sporulation and 61. Perrakis A, Sixma TK, Wilson KS, Lamzin VS (1997) wARP: Improvement and extension degradative enzyme production. Proteins 79:2566–2577. of crystallographic phases by weighted averaging of multiple-refined dummy atomic 34. Angus-Hill ML, Dutnall RN, Tafrov ST, Sternglanz R, Ramakrishnan V (1999) Crystal models. Acta Crystallogr D Biol Crystallogr 53:448–455. structure of the histone acetyltransferase Hpa2: A tetrameric member of the Gcn5- 62. Kabsch W (1976) Crystal physics, diffraction, theoretical and general crystallography. related N-acetyltransferase superfamily. J Mol Biol 294:1311–1325. Acta Crystallogr A 32:922–923. 35. Leslie AG, Moody PC, Shaw WV (1988) Structure of chloramphenicol acetyltransferase 63. DeLano WL (2002) The PyMOL Molecular Graphics System (DeLano Scientifc, San at 1.75-A resolution. Proc Natl Acad Sci USA 85:4133–4137. Carlos, CA). 36. Vetting MW, et al. (2005) Structure and functions of the GNAT superfamily of ace- 64. Gille C, Frömmel C (2001) STRAP: Editor for structural alignments of proteins. tyltransferases. Arch Biochem Biophys 433:212–226. Bioinformatics 17:377–378.

11674 | www.pnas.org/cgi/doi/10.1073/pnas.1206828109 Han et al. Downloaded by guest on September 23, 2021