International Journal of Molecular Sciences

Review Structure and Functional Diversity of GCN5-Related N-Acetyltransferases (GNAT)

Abu Iftiaf Md Salah Ud-Din 1, Alexandra Tikhomirova 1 and Anna Roujeinikova 1,2,*

1 Infection and Immunity Program, Monash Biomedicine Discovery Institute; Department of Microbiology, Monash University, Clayton, Victoria 3800, Australia; [email protected] (A.I.M.S.U.-D.); [email protected] (A.T.) 2 Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia * Correspondence: [email protected]; Tel.: +61-3-9902-9194; Fax: +61-3-9902-9222

Academic Editor: Claudiu T. Supuran Received: 30 May 2016; Accepted: 20 June 2016; Published: 28 June 2016

Abstract: General control non-repressible 5 (GCN5)-related N-acetyltransferases (GNAT) catalyze the transfer of an acyl moiety from acyl coenzyme A (acyl-CoA) to a diverse group of substrates and are widely distributed in all domains of life. This review of the currently available data acquired on GNAT enzymes by a combination of structural, mutagenesis and kinetic methods summarizes the key similarities and differences between several distinctly different families within the GNAT superfamily, with an emphasis on the mechanistic insights obtained from the analysis of the complexes with substrates or inhibitors. It discusses the structural basis for the common acetyltransferase mechanism, outlines the factors important for the substrate recognition, and describes the mechanism of action of inhibitors of these enzymes. It is anticipated that understanding of the structural basis behind the reaction and substrate specificity of the enzymes from this superfamily can be exploited in the development of novel therapeutics to treat human diseases and combat emerging multidrug-resistant microbial infections.

Keywords: GNAT; acetyltransferase; crystal structure; reaction mechanism; enzyme inhibitor; catalytic residues

1. Introduction The acylation reactions, catalyzed by diverse groups of enzymes, play an important role in numerous biological processes. The focus of this review is the structure/activity relationships in the superfamily of general control non-repressible 5 (GCN5)-related N-acetyltransferases (GNAT, the term introduced into classification by Neuwald and Landsman in 1997 [1]), with an emphasis on the mechanistic insights obtained from the analysis of the GNAT complexes with substrates or inhibitors. GNAT enzymes catalyze the transfer of an acyl group from acyl coenzyme A (acyl-CoA) to an amino group of a wide range of substrates. Although most GNAT enzymes use acetyl-CoA (AcCoA), there are GNAT families that prefer different acyl donors such as myristoyl CoA or succinyl CoA [2–4]. The first two three-dimensional structures of representatives of this superfamily, reported in 1998, were those of aminoglycoside N-acetyltransferase from multidrug-resistant Serratia marcescens, determined by Wolf and co-workers [5], and histone acetyltransferase 1 (HAT1) from Saccharomyces cerevisiae, solved by Dutnall [6]. Over the recent decades, more than 309,000 members of the GNAT superfamily have been identified in all domains of life. There are at least 272,000 GNAT proteins in bacteria (of which 118,000 are found in Proteobacteria), 31,000 proteins in eukaryotes and 6500 proteins in archaea [7]. These enzymes are involved in diverse cellular processes, including stress regulation, transcription control, maintenance of a reducing state of the cytosol, protection of cellular contents against

Int. J. Mol. Sci. 2016, 17, 1018; doi:10.3390/ijms17071018 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2016, 17, 1018 2 of 45 oxidants, development of antibiotic resistance and detoxification of thio-reactive compounds [8–10]. GNAT enzymes also have anabolic and catabolic functions in both prokaryotes and eukaryotes [2,8]. For example, glucosamine-6-phosphate N-acetyltransferase-1 of S. cerevisiae is involved in the biosynthesis of a metabolite, uridine diphosphate (UDP)-N-acetylglucosamine [11], whereas spermidine/spermine-N1-acetyltransferase plays an important role in polyamine catabolism [12].

2. Overall Architecture of General Control Non-Repressible 5 (GCN5)-Related N-Acetyltransferase (GNAT) Superfamily Enzymes Structural information is currently available for members of 17 distinct families within the GNAT superfamily. Their respective names, Enzyme Commission (EC) classification, source organisms, substrates and the Protein Data Bank (PDB) codes are listed in Table1. The characteristic conserved core fold of the GNAT superfamily proteins consists of six to seven β-strands and four α-helices, which are connected in order β0-β1-α1-α2-β2-β3-β4-α3-β5-α4-β6 and arranged in the topology shown in Figure1. GNAT enzymes show moderate pairwise sequence identity (3%–23%) [10]. They vary significantly at the C-terminus while the N-terminus is relatively conserved. Their amino acid sequences harbor four conserved motifs (A–D), which are arranged in order C-D-A-B [2]. Motifs C and D play an important role in maintaining the protein’s stability, while motifs A and B contain residues involved in acyl-CoA and acceptor substrate binding, respectively. Generally, the N-terminal β1 and β2 strands are connected by a loop incorporating two helices (α1 and α2). However, the length, number and position of α-helices in this loop show significant variations across different GNAT enzymes. For example, the second helix (α2) is missing in the structure of yeast HAT1 [6], while aminoglycoside 61-N-acetyltransferase from Enterococcus faecium has an additional α-helix between the strands β1 and β2 [13]. The significant variation in this region of the GNAT enzymes allows formation of different acceptor substrate binding sites and thus reflects variation in their substrate specificity. The β4 strand of motif A is parallel to the short β5 strand of motif B; these two strands create a V-shaped opening in the center of the catalytic domain of the protein to accommodate acyl-CoA [2]. Motif B residues are not well conserved across the entire GNAT superfamily. However, within individual families, the enzymes have many common residues within this region. Together with loop β1β2, the α4 helix of motif B and strand β6 at the C-terminal end form the binding site for the acceptor substrate. The structural variations in this region allow different GNAT proteins to recognize a diverse group of acceptor substrates. Most GNAT enzymes have a β-bulge at the center of strand β4 next to the end of the short parallel β5 strand. The β-bulge generates an oxyanion hole that contributes to the stabilization of the tetrahedral reaction intermediate [3,14]. Another distinctive conserved feature is the pyrophosphate binding site in the loop N-terminal to the α3 helix of motif A. Strand β4, helix α3 and strand β5 form a βαβ motif similar to that of the nucleotide-binding Rossman fold [15]. The signature motif at the pyrophosphate binding site, referred to as the “P-loop”, is made up of six amino acids, the amides of which form hydrogen bonds with the phosphate oxygen atoms of acyl-CoA. The consensus “P-loop” sequence in GNAT enzymes is Gln/Arg-x-x-Gly-x-Gly/Ala, where x is any amino acid [5,10].

2.1. Aminoglycoside N-Acetyltransferases Family (AAC, EC 2.3.1.81) Aminoglycoside N-acetyltransferases (AACs) found in bacteria catalyze regioselective transfer of the acetyl group to one of the four amino groups present on the aminocyclitol ring of a wide variety of aminoglycoside antibiotics (Figure2), including apramycin, gentamicin, kanamycin, neomycin and tobramycin. Bacteria that possess or acquire the gene encoding AAC show resistance to aminoglycosides because acetylated aminoglycosides have a lower affinity for the tRNA binding site on the bacterial 30S ribosomal subunit [16]. AACs are classified into four major classes based on the site of acetylation: AAC(1), AAC(21), AAC(3) and AAC(61)[17]. Recently, a novel type of aminoglycoside modifying enzyme, named enhanced intracellular survival (Eis) protein, was identified in Mycobacterium sp. Eis acetylates multiple amino groups of aminoglycosides and thus confers Int. J. Mol. Sci. 2016, 17, 1018 3 of 45

resistanceInt. J. Mol. to aSci. wide 2016, 17 range, 1018 of aminoglycoside antibiotics [18–20]. Structural information is available3 of 44 on sevenInt. different J. Mol. Sci. 2016 aminoglycoside-modifying, 17, 1018 enzyme subfamilies: AAC(3)-Ia, AAC(21)-Ic, AAC(63 of 44 1)-Ib, AAC(6AAC(61)-Ie,′)-Ib, AAC(6 AAC(61)-Ii,′)-Ie, AAC(6 AAC(61)-Iy′)-Ii, and AAC(6 Eis [5′)-Iy,10, 19and,21 Eis–26 [5,10,19,21–26],], with a representative with a representative from each subfamilyfrom AAC(6′)-Ib, AAC(6′)-Ie, AAC(6′)-Ii, AAC(6′)-Iy and Eis [5,10,19,21–26], with a representative from describedeach subfamily below. described below. each subfamily described below.

FigureFigure 1. Topology 1. Topology of the of general the general control control non-repressible non-repressible 5 (GCN5)-related 5 (GCN5)-relatedN-acetyltransferase N-acetyltransferase (GNAT) Figure 1. Topology of the general control non-repressible 5 (GCN5)-related N-acetyltransferase enzymes.(GNAT) The enzymes. secondary Thestructure secondary elements structure elements encompassing encompassing the conserved the conserved sequence sequence motifs motifs C (β C1– α1), (GNAT)(β1–α1), enzymes.D (β2–β3), The A ( secondaryβ4–α3) and structure B (β5–α4) elements are colored encompassing green, blue, the re conservedd and magenta, sequence respectively. motifs C D(β2–β3), A (β4–α3) and B (β5–α4) are colored green, blue, red and magenta, respectively. The location (Theβ1– αlocation1), D (β 2–of βthe3), Aconserved (β4–α3) and“P-loop” B (β5– (αβ4–4) αare3) coloredis shown. green, The blue,least conservedred and magenta, secondary respectively. structure of the conserved “P-loop” (β4–α3) is shown. The least conserved secondary structure elements (strands Theelements location (strands of the β 0conserved and β6 and “P-loop” helix α2), (β that4–α 3)are is absent shown. in Thesome least GNAT conserved proteins, secondary are colored structure wheat. β0 andelementsβ6 and (strands helix α β2),0 and that β6 are and absent helix α in2), somethat are GNAT absent proteins, in some GNAT are colored proteins, wheat. are colored wheat.

Figure 2. Chemical structure of an aminoglycoside antibiotic (ribostamycin) showing the central FigureFigureaminocyclitol 2. Chemical 2. Chemical ring structure and structure acetyl of group of an an aminoglycoside modificationaminoglycoside sites antibiotic antibiotic(1, 2′, 3 and (ribostamycin)(ribostamycin) 6′). showing showing the central the central aminocyclitol ring and acetyl group modification sites (1, 2′, 3 and 6′). aminocyclitol ring and acetyl group modification sites (1, 21, 3 and 61). Aminoglycoside 2-N-acetyltransferase-Ic of Mycobacterium tuberculosis (MtAAC(2′)-Ic) can performAminoglycoside both N- and 2-ON-acetylation-acetyltransferase-Ic of the 2 ′ ofamino Mycobacterium group of a tuberculosiswide range (MtAAC(2 of aminoglycoside′)-Ic) can 1 Aminoglycosideperformantibiotics both [22]. N MtAAC(2- and 2-N O-acetyltransferase-Ic-acetylation′)-Ic is a 20.0 of kDa the protein 2′ ofamino encodedMycobacterium group by of the a chromosomalwide tuberculosis range ofRv0262c(MtAAC(2 aminoglycoside gene [23].)-Ic) can 1 performantibioticsIt is a both dimer [22].N in- the and MtAAC(2 crystalO-acetylation (Figure′)-Ic is a 3A). 20.0 of Structural kDa the protein 2 aminoanalysis encoded groupof the by MtAAC(2 the of chromosomal a wide′)-Ic ternary range Rv0262c ofcomplexes aminoglycoside gene with[23]. antibioticsItCoA is a anddimer [22 aminoglycosides]. in MtAAC(2 the crystal1)-Ic (Figure revealed is a 20.03A). that Structural kDa MtAAC(2 protein analysis′)-Ic encoded has of thea β byMtAAC(2-bulge the chromosomalin the′)-Ic β ternary4 strand complexesRv0262c (residues genewith G83 [23]. It is aCoAand dimer V84) and in andaminoglycosides the a crystalV-shaped (Figure cleft revealed between3A). Structuralthat the MtAAC(2 β4 and analysis β′)-Ic5 strands, has of a the β that-bulge MtAAC(2 serves in the as1 )-Icβthe4 strand AcCoA ternary (residues binding complexes G83site. with and V84) and a V-shaped cleft between the β4 and β5 strands, that serves as the AcCoA binding site. CoAMtAAC(2 and aminoglycosides′)-Ic has an atypical revealed “P-loop”, that MtAAC(2 the sequence1)-Ic hasof which a β-bulge (G92-Q93-R94-L95-V96) in the β4 strand (residuesdoes not G83 MtAAC(2′)-Ic has an atypical “P-loop”, the sequence of which (G92-Q93-R94-L95-V96) does not and V84)match and the aconsensus V-shaped found cleft in between other GNAT the β proteins4 and β. 5The strands, “P-loop” that interacts serves with as the the AcCoA pyrophosphate binding site. match the consensus found in other GNAT proteins. The “P-loop” interacts with the pyrophosphate MtAAC(2arm of1)-Ic CoA has via an both atypical direct “P-loop”,and water-mediated the sequence hydrogen of which bonds (G92-Q93-R94-L95-V96) [23]. The backbone amide does group not of match armV84 offorms CoA a via hydrogen both direct bond and with water-mediated the carbonyl hy oxygendrogen of bonds AcCoA [23]. and The is ba thoughtckbone amideto stabilize group the of the consensus found in other GNAT proteins. The “P-loop” interacts with the pyrophosphate arm of V84tetrahedral forms aintermediate hydrogen bond formed with during the carbonylthe acetyl oxygen transfer of reaction AcCoA [23]. and The is hydrogenthought to bond stabilize between the CoAtetrahedralthe via backbone both direct intermediate amide and group water-mediated formed of G83 during and thethe hydrogen acetyl3 amino transfer bondsgroup reaction of [23 the]. Thesubstrate[23]. backboneThe hydrogenis important amide bond for group between proper of V84 formsthepositioning a hydrogenbackbone of amide bondthe acceptor withgroup the substrateof carbonylG83 and for the oxygenthe 3 direct amino of nucleophilic AcCoA group of and the attack. is substrate thought The hydroxylis to important stabilize group thefor ofproper tetrahedral Y126 intermediatepositioningis ~3.6 Å formedaway of the from during acceptor the sulfur the substrate acetyl moiety transfer for of the CoA direct reaction and nucleophiliccould [23 ].serve The asattack. hydrogen the general The bondhydroxyl acid between during group catalysis, the of Y126 backbone amideiswhile group~3.6 theÅ ofaway E82 G83 orfrom andW181 the the weresulfur 3 amino suggested moiety group of toCoA of act the and as substrate thecould remote serve is importantgeneral as the generalbase for via properacid well-ordered during positioning catalysis, water of the acceptorwhilemolecules substrate the E82[23]. foror W181 the direct were nucleophilicsuggested to attack.act as the The remote hydroxyl general group base of via Y126 well-ordered is ~3.6 Å awaywater from molecules [23]. the sulfur moiety of CoA and could serve as the general acid during catalysis, while the E82 or W181 were suggested to act as the remote general base via well-ordered water molecules [23].

Int. J. Mol. Sci. 2016, 17, 1018 4 of 45

Structural analysis of the plasmid-encoded aminoglycoside 3-N-acetyltransferase of S. marcescens (SmAAC(3), 168 aa) in complex with CoA revealed that SmAAC(3) forms a dimer in the crystal [5]. SmAAC(3) has a β-bulge in the β4 strand (residue Y109 and D110) and a conserved “P-loop” R118-R119-Q120-G121-I122-A123 that interacts with the diphosphate moiety of CoA. The strands β4 and β5 are splayed apart to form the CoA binding site (Figure3B). It was shown that a homolog of SmAAC(3), gentamicin 3-N-acetyltransferase from Pseudomonas aeruginosa, follows a direct acetyl transfer mechanism [27]. Kinetic analysis of gentamicin 3-N-acetyltransferase showed that a bisubstrate analog, produced by covalent linking of the reaction product (3-N-chloroacetylgentamicin) to CoA, inhibits this enzyme in vitro with high specificity [27]. The 196-residue Escherichia coli AAC(61)-Ib (EcAAC(61)-Ib) is a chromosome-encoded aminoglycoside-modifying enzyme that confers bacterial resistance to the antibiotics amikacin, 1 1 kanamycin and tobramycin [25,26,28]. The AAC(6 )-Ib11 of Salmonella enterica (SeAAC(6 )-Ib11), a close homolog of EcAAC(61)-Ib, confers resistance to a broader range of aminoglycosides that include 1 1 amikacin and gentamicin [29]. EcAAC(6 )-Ib is a monomer in solution [25], while SeAAC(6 )-Ib11 exists as a mix of monomers and dimers [30]. The crystal structure of EcAAC(61)-Ib in complex with AcCoA and kanamycin C revealed that the active site is located within the monomer (Figure3C) [ 25]. 1 1 The active-site residues equivalent to Q106 and L107 of EcAAC(6 )-Ib are substituted in SeAAC(6 )-Ib11 with leucine and serine, respectively, which results in a wider substrate binding site that can accommodate larger aminoglycoside antibiotics. The EcAAC(61)-Ib homolog from P. aeruginosa follows an ordered sequential kinetic mechanism where AcCoA binds to the active site first, followed by the aminoglycoside substrate. The EcAAC(61)-Ib structure contains conserved features such as the “P-loop” L124-G125-K126-G127-L128-G129 and the V-shaped AcCoA binding cleft [25]. Differences with other members of the GNAT superfamily include the additional β7 strand that is placed between the β5 and β6 strands and a lack of the β-bulge. Detailed analysis of the EcAAC(61)-Ib structure suggested that the essential residue D115 [25,31] could act as a general base to accept a proton in the AAC-catalyzed reaction, while the main-chain amide of Q116 forms a hydrogen bond with the carbonyl group of AcCoA, which is likely to stabilize the tetrahedral intermediate. The semi-conserved Y164 was proposed to act as a general acid either directly or via a conserved water molecule [25]. Recently, Chiem and colleagues [32] reported identification of an EcAAC(61)-Ib inhibitor (1-[3-(2-aminoethyl)benzyl]-3-piperidin-1-ylmethyl)pyrrolidin-3-ol) by an in silico docking approach. The inhibitor targets the kanamycin A binding site. In addition, this compound could prevent the growth of an amikacin-resistant Acinetobacter baumannii clinical strain, suggesting that it also inhibits A. baumannii AAC(61)-Ib [32]. The aminoglycoside 61-N-acetyltransferase-Ie of Staphylococcus warneri (SwAAC(61)-Ie-APH(211)) is a bifunctional enzyme which has an N-terminal N-acetyltransferase domain (AAC) and a C-terminal aminoglycoside phosphotransferase (APH) domain [24]. It confers resistance to nearly all commonly prescribed aminoglycoside antibiotics [26]. The 179-residue recombinant acetyltransferase domain crystallizes as a monomer. Structural analysis of this domain in complex with a sulfinic acid form of CoA showed that the CoA molecule is bound at the conserved V-shaped cleft formed by two β-strands splayed apart [24] (Figure3D). SwAAC(6 1)-Ie-APH(211) has no β-bulge at the active site and possesses an atypical glycine rich “P-loop” W108-S109-K110-G111-I112-G113 that interacts with the diphosphate arm of CoA. The aminoglycoside binding site is located in a highly negatively charged pocket formed by residues from the β4, β5 and β6 strands [24]. The chromosomally encoded 20.7 kDa 61-N-acetyltransferase-Ii from E. faecium (EfAAC(61)-Ii) has a broad substrate specificity for aminoglycosides [33]. In addition, it can acetylate histones and other small basic proteins [13]. The EfAAC(61)-Ii is a dimer both in solution and in the crystal [34]. Structural analysis revealed that EfAAC(61)-Ii has a β-bulge in strand β4 (residue H74 and P75), an atypical “P-loop” R83-K84-N85-Q86-I87-G88 and a V-shaped binding site between the β4 and β5 strands which are splayed apart to accommodate AcCoA (Figure3E). Like EcAAC(6 1)-Ib and 1 1 SeAAC(6 )-Ib11, EfAAC(6 )-Ii has an additional strand (β7) located between the β5 and β6 strands. Int. J. Mol. Sci. 2016, 17, 1018 5 of 45

The hydroxyl group of Y147 forms a hydrogen bond with the sulfur atom of AcCoA. Kinetic analysis of EfAAC(61)-Ii variants showed that Y147 does not act as a general acid in catalysis; instead, it is thought to be important for maintaining an optimal orientation of the acetyl group for efficient transfer [35]. EfAAC(61)-Ii follows an ordered Bi-Bi mechanism (two on, two off) in which AcCoA binds to the enzyme first, followed by the aminoglycoside substrate [35,36]. Aminoglycoside-CoA bisubstrate analogs inhibit EfAAC(61)-Ii at nanomolar concentrations, with some of them showing antimicrobial activity both in vitro and in vivo [37,38]. Analysis of their structure-activity relationships revealed that the inhibitory activity does not require the presence of the adenosine diphosphate (ADP) moiety, or the second or third aminoglycoside ring. At least one phosphate group is needed, although the pyrophosphate moiety can be replaced with β-dicarbonyl groups [37,38]. The chromosomally encoded aminoglycoside 61-N-acetyltransferase-Iy from S. enterica (SeAAC(61)-Iy) can catalyze 61-N-acetylation of a wide range of 4,6- and 4,5-disubstituted aminoglycosides [22]. The 145-amino-acid SeAAC(61)-Iy is a dimer both in solution and in the crystal [39]. It has a β-bulge in the β4 strand (residues E79 and G80), a typical “P-loop” (R88-Q89-R90- G91-V92-A93) and a V-shaped AcCoA binding site. The dimer interface has an exchanged β6 strand (Figure3F)—a feature also found in S. cerevisiae histone N-acetyltransferase. Structural analysis of the SeAAC(61)-Iy/CoA/ribostamycin ternary complex revealed that the 61 amino group of ribostamycin is positioned close to the sulfur atom (~3.5 Å) of CoA, which would favor the direct acetyl transfer reaction. D115 on the β5 strand forms a water-mediated hydrogen bond with the 61-amino group of the antibiotic molecule and was therefore proposed to serve as a general base in catalysis. No specific residue that could act as a general acid in this reaction was identified; a water-mediated protonation mechanism was proposed instead [39]. It follows a random sequential, rather than ordered, kinetic mechanism [22]. Structural and kinetic analysis of SeAAC(61)-Iy showed that aminoglycoside-CoA bisubstrate analogs inhibit the enzyme at micromolar concentrations by binding to its active site in a mode similar to that observed for CoA and ribostamycin in the crystal structure of the SeAAC(61)-Iy-CoA-ribostamycin complex [22]. Eis is a 45 kDa protein that enhances survival of Mycobacterium sp. within host cells by suppressing the host defense mechanisms [19,20]. Unlike other AACs, Eis and its homologs can acetylate multiple amines of aminoglycosides [19,40,41]. For example, Eis can transfer an acetyl group both on the 61- and 3-amine group of kanamycin, and on the 3- and 4-amino-2-hydroxybutyryl group of amikacin [19]. In addition, Eis can use peptides as substrates. Eis exists as a hexamer both in solution and in the crystal [42], where two three-fold symmetrical trimers assemble into an asymmetric “sandwich”. Structural analysis of Eis in complex with CoA and acetylated hygromycin revealed that each Eis monomer harbors an N-terminal GNAT domain (residue 1–150), a central GNAT domain (residue 151–291) and a C-terminal domain with an atypical fold of the animal sterol carrier protein lacking a hydrophobic cavity (residues 292–402) (Figure3G). Only the N-terminal GNAT domain is catalytically active; the central, inactive, GNAT domain lacks the conserved Gln/Arg residue in the “P-loop” that in other GNAT enzymes participates in recognition of the CoA pyrophosphate moiety. Aminoglycosides bind in the negatively charged pocket formed between the N-terminal and central GNAT domains [42]. The main-chain amino group of H119 plays an important role in proper positioning of the acceptor substrate, while the carbonyl oxygen of AcCoA is hydrogen-bonded with both F84 and V85, which might contribute to the stabilization of the tetrahedral reaction intermediate [42]. F402 could serve as a remote base that extracts a proton from the amino group of the acceptor substrate via a water molecule. The conserved Y126 residue is ~3.4 Å away from the sulfhydryl group of CoA and could serve as a general acid that donates a proton to the leaving thiolate anion. A mutagenesis study confirmed the presence of one catalytically active GNAT domain in Eis and the role of the Y126 residue as a general acid [42]. Comparison of the crystal structures of Eis proteins from M. tuberculosis (MtEis) and Mycobacterium smegmatis (MsEis) [41] revealed that the substrate binding site of MtEis is located in a deep narrow channel, where MsEis has a deep round pocket [41]. This is consistent with the endo-type peptide acylation activity of MtEis (on the Lys side chain) and the exo-type activity of MsEis (on the N-terminal amino group of peptides). Int. J. Mol. Sci. 2016, 17, 1018 6 of 45 Int. J. Mol. Sci. 2016, 17, 1018 6 of 44

Figure 3. 3. Cont.Cont.

Int. J. Mol. Sci. 2016, 17, 1018 7 of 45 Int. J. Mol. Sci. 2016, 17, 1018 7 of 44

FigureFigure 3.3. CartoonCartoon representation representation of ofthe thestructures structures of aminoglycoside of aminoglycoside N-acetyltransferases.N-acetyltransferases. (A) 1 (AAminoglycoside) Aminoglycoside 2′-N 2-acetyltransferase-Ic-N-acetyltransferase-Ic from from MycobacteriumMycobacterium tuberculosis tuberculosis in complexin complex with CoA with and CoA andribostamycin ribostamycin (Rib) (Rib) (PDB (PDB ID: ID:1M4G 1M4G [23]); [23 ]);(B) ( Baminoglycoside) aminoglycoside 3- 3-N-acetyltransferase-IaN-acetyltransferase-Ia from from SerratiaSerratia 1 marcescensmarcescens inin complexcomplex withwith AcCoAAcCoA (PDB ID: 1BO4 [5]); [5]); ( C)) aminoglycoside aminoglycoside 6 6′-N-N-acetyltransferase-Ib-acetyltransferase-Ib fromfrom EscherichiaEscherichia coli coli inin complex complex with with AcCoA AcCoA and and kanamycin kanamycin C (KNC) C (KNC) (PDB (PDB ID: 1V0C ID: 1V0C [25]); [(25D)]); 1 (Daminoglycoside) aminoglycoside 6′-N 6-acetyltransferase-Ie-N-acetyltransferase-Ie from fromStaphylococcusStaphylococcus warneri warneri complexcomplex with a with sulfinic a sulfinic acid acidform form of coenzyme of coenzyme A (CoA) A (CoA) and kanamycin and kanamycin A (KAN) A (KAN) (PDB (PDBID: 4QC6 ID: 4QC6[24]); ( [E24) ]);aminoglycoside (E) aminoglycoside 6′-N- 1 6acetyltransferase-Ii-N-acetyltransferase-Ii from Enterococcus from Enterococcus faecium in faecium complexin with complex CoA (PDB with ID: CoA 1N71 (PDB [34]); ID: (F) 1N71Salmonella [34]); 1 (Fenterica) Salmonella aminoglycoside enterica aminoglycoside 6′-N-acetyltransferase-Iy 6 -N-acetyltransferase-Iy in complex with in complex CoA and with ribostamycin CoA and ribostamycin (Rib) (PDB (Rib)ID: 1S3Z (PDB [39]); ID: 1S3Zand ( [G39) ]);M. and tuberculosis (G) M.tuberculosis enhanced intracellularenhanced intracellular survival (Eis) survival in complex (Eis)in with complex CoA and with CoAtobramycin and tobramycin (PDB ID: (PDB4JD6 [19]). ID: 4JD6 The[ conserved19]). The conserved and non-conserved and non-conserved motifs are motifscolored are as coloredin Figure as 1 in Figure(motif1 C—green,(motif C—green, motif D—blue, motif D—blue, motif A—red, motif A—red, motif motifB—magenta, B—magenta, non-conserved non-conserved N-terminal N-terminal and andC-terminal C-terminal regions—wheat). regions—wheat). The C-te Therminal C-terminal animal animal sterol sterol carrier carrier domain domain of Eis ofis Eiscolored is colored cyan. The cyan. TheAcCoA/CoA AcCoA/CoA cofactor cofactor is drawn is drawn as black as black sticks, sticks, wh whereasereas the the substrates substrates (tobramycin (tobramycin and and kanamycin) kanamycin) areare shownshown inin blackblack usingusing ball-and-stickball-and-stick representation.

Int. J. Mol. Sci. 2016, 17, 1018 8 of 45

2.2. Histone N-Acetyltransferase Family (HATs, EC 2.3.1.48) Histone N-acetyltransferases (HATs) acetylate the side chains of the conserved lysine residues on the N-terminal tail of histone proteins to promote transcriptional activation. HATs play a crucial role in chromatin remodeling and gene expression in eukaryotes [43]. HATs are classified into two types based on their subcellular localization and substrate specificities: (i) type A HATs which are present only in the nucleosome and can modify histones that are incorporated into chromatin; and (ii) type B HATs which are present in both the cytoplasm and nucleosome, and can acetylate only soluble free histones [43,44]. Histone N-acetyltransferase 1 (HAT1), found in S. cerevisiae and human, is type B HAT that acetylates the K5 and/or K12 residue of soluble free histone H4 [6,45]. Acetylation of histones by HAT1 might play an important role in chromatin assembly and DNA repair [44,46]. Several studies reported that there is a link between HAT1 expression levels and cancer [44]. Structural analysis of the ternary complex of human HAT1 (hHAT1) with AcCoA and histone H4 peptide revealed that hHAT1 has three domains: an N-terminal domain (residues 23–136), a central GNAT domain (residues 137–270), and a C-terminal domain (residues 271–341) (Figure4A). The AcCoA binds in the cleft between the central and C-terminal domains [45]. The central domain has a glycine-rich “P-loop” that interacts with the pyrophosphate arm of AcCoA. The acetyl moiety of AcCoA is anchored by the interactions with I186, P278 and Y282 such that the acetyl group is placed close to the side chain of K12 of the histone H4 peptide, with a distance of ~4.3 Å between its carbonyl carbon and the ε-amino group of K12 [45]. The histone peptide is accommodated between the central and N-terminal domains. The conserved residues E64 and T199 of the N-terminal domain play an important role in the recognition of the acceptor substrate of hHAT1 [45]. Mutagenesis data confirmed that the combined action of the active-site residues E187, E276 and D277 deprotonates the ε-amino group of reactive K12 of the histone 4 peptide via a water molecule to facilitate the direct nucleophilic attack on the thioester acetate of AcCoA [45]. The structural homolog of hHAT1 in S. cerevisiae (ScHAT1) has an almost identical overall structural topology, except ScHAT1 has a longer β12–β13 loop, and a shorter β12 strand and β11–β12 loop [6]. The S. cerevisiae Esa1 (ScEsa1), type A HAT, is the sole histone acetyltransferase expressed in budding yeast [47]. ScEsa1 can acetylate histones H4, H2A and H2AZ [48–53]. It plays an important role in the regulation of gene expression and DNA repair [54–56]. The 445-amino-acid ScEsa1 has a central GNAT domain (residue 272–329) flanked by an N-terminal and C-terminal subdomains (Figure4B) [ 57,58]. Structural analysis of ScEsa1 in complex with AcCoA showed that motif D is missing, and that AcCoA binds between the core GNAT domain and the C-terminal subdomain. Mutagenesis studies and structural analysis of a ScEsa1 complex with a bisubstrate inhibitor H4K16CoA, in which CoA is covalently linked to the side-chain amino group of the acetyl lysine residue in the histone H4 peptide substrate, revealed that autoacetylation of a conserved lysine (K262) in ScEsa1 contributes to substrate binding and acetylation [59]. Autoacetylation of ScEsa1 is also essential for the viability of yeast cells. Detailed analysis of the active site of ScEsa1 showed that E338 (equivalent to E579 in hPCAF and E255 in ScHat1) could act as a remote general base to extract a proton from the acceptor substrate [60]. Mutagenesis study confirmed its role in the acetyl transfer reaction [58]. Although the observation of an acetyl-cysteine covalent adduct in the crystals of ScEsa1 previously led to a hypothesis that this enzyme may employ a ping-pong reaction mechanism [57], subsequent kinetics analysis disproved that hypothesis and showed that ScEsa1 follows a direct nucleophilic attack mechanism [60]. Histone acetyltransferase of S. cerevisiae GCN5 (ScGCN5) is type A HAT that preferentially acetylates histones H3 (on K14) and H4 (on K8 and K16), but can also acetylate non-histone chromatin proteins including Spt2 [61]. ScGCN5 plays an important role in the control of expression of cell cycle-related and apoptosis-related genes [62]. It was first identified as an essential gene for biosynthesis of amino acids in yeast [63]. In addition, the GCN5 null mouse showed increased apoptosis and mesodermal defects during embryo development [64]. Structural analysis revealed Int. J. Mol. Sci. 2016, 17, 1018 9 of 45 that ScGCN5 has a β-bulge in strand β4 (A124 and F125) and a conserved “P-loop” in motif A (Q133-V134-R135-G136-Y137-G138) that contributes to the AcCoA binding (Figure4C) [ 65]. Structural analysis suggested, and mutagenesis and kinetics studies confirmed, that a conserved glutamate residue (E173) at the bottom of the putative substrate binding cleft serves as a general base to extract a proton from the ε-NH2 group of the lysine side chain on the substrate [65]. Structures of GCN5 homologs from other sources were subsequently solved, including those from human [66] and Tetrahymena thermophila [67–70]. Analysis of T. thermophila GCN5 (TtGCN5) in free form, in complex with AcCoA and in complex with CoA and a histone H3 peptide revealed a conserved sequence motif (G-K-X-P) that determines the histone-binding specificity [67–70]. The backbone NH group of L126 forms a hydrogen bond with the carbonyl oxygen of the acetyl group of AcCoA. L126 is therefore thought to play a crucial role in the reaction mechanism by polarizing the carbonyl group of the thioester and stabilizing the reaction intermediate [68]. TtGCN5’s conserved residue E122 (equivalent to residue E173 of ScGCN5) might act as a general base via a well-ordered water molecule to facilitate the direct acetyl transfer [67,68]. In the human homolog, the E575 residue, equivalent to residue E173 of ScGCN5, could serve as a general base to deprotonate the substrate [66]. Interestingly, the crystal structure of TtGCN5 bound to a peptide-CoA bisubstrate inhibitor suggested that after lysine acetylation, the H3 histone product is displaced from the substrate binding site through a rearrangement within the C-terminal domain of the enzyme [69]. The human p300/CBP-associating factor (hPCAF) facilitates activation of transcription by acetylating histone proteins in the nucleosome and other transcriptional activators, including the tumor protein p53 [71]. hPCAF (832 aa) is type B HAT that mainly acetylates K14 of histone H3. It also specifically acetylates p53 at K320 to promote its response to cellular DNA damage [72]. Structural and biochemical analysis of hPCAF in complex with CoA revealed that hPCAF exists as a dimer both in solution and in the crystal [72,73]. It has three domains: an N-terminal domain with limited homology to mammalian GNAT enzymes (1–492) [74], a central, catalytic HAT domain (residues 493–653) that shares a high degree of sequence homology with other GNAT histone acetyltransferases, and a C-terminal bromodomain (residues 725–819) (Figure4D). The catalytic domain has a “P-loop” Q581-V582-K583-G584-Y585-G586 and a β-bulge in the β4 strand (residues V572 and F573). The CoA is bound in the V-shaped cleft between the β4 and β5 strands. Detailed analysis of the substrate binding site revealed that E570 (equivalent to E173 in ScGCN5) is optimally positioned to serve as a general base to extract a proton from the lysine residue of the substrate via a water molecule. The main-chain amide nitrogen of C574 forms a hydrogen bond with the carbonyl oxygen of AcCoA, that could stabilize the tetrahedral reaction intermediate [73]. The S. cerevisiae Hpa2 (ScHpa2) is type A HAT that acetylates the ε-amino group of lysine residues of histones H3 (K4 and K14) and H4 (K5, K12 and K8) [75]. In addition, ScHpa2 can acetylate non-histone chromosomal proteins (e.g., non-histone proteins Nhp6A and Nhp6B, high mobility group proteins Hmo1 and Hmo2) and polyamines, including putrescine, spermine and spermidine [75]. ScHpa2 (156 aa) exists as a dimer in solution. However, the ScHpa2/AcCoA binary complex is tetrameric both in solution and in the crystal [76]. This tetramer is a dimer of dimers, in which the adenine group of the AcCoA bound to one dimer interacts with the adenine group of the second AcCoA molecule bound to another dimer. The two ScHpa2 monomers assemble into a strand-swapped dimer shown in Figure4E, where the β7 strand of each monomer is inserted between the β5 and β6 strands of the opposite monomer. ScHpa2 has a β-bulge in the β4 strand (residues N91 and D92) and an atypical “P-loop” V101-K102-G103-A104-G105-G106. Y139, the hydroxyl group of which forms a hydrogen bond with the sulfur atom of AcCoA, was proposed to serve as a general acid in the reaction. Mutagenesis of the equivalent Tyr residue (Y168) in spermidine/spermine N1-acetyltransferase (SSAT) confirmed its role in catalysis [77]. The positive charge around the entrance to the active site of ScHpa2 favors a deprotonated state of the ε-amino group of the substrate, thus negating a requirement for a specific general base residue [76]. Int. J. Mol. Sci. 2016, 17, 1018 10 of 45 Int. J. Mol. Sci. 2016, 17, 1018 10 of 44

FigureFigure 4. 4.Cartoon Cartoonrepresentation representation of of the the structures structures of of histone histoneN N-acetyltransferases.-acetyltransferases. ( A(A)) Human Human HAT1 HAT1 in complexin complex with with AcCoA AcCoA and and a histone a histone H4 peptideH4 peptide (PDB (PDB ID: 2P0WID: 2P0W [45]); [45]); (B) yeast(B) yeast Esa1 Esa1 in complex in complex with AcCoAwith AcCoA (PDB ID:(PDB 1MJB ID: [1MJB57]); (C[57]);) yeast (C) GCN5 yeast GCN5 acetyltransferase acetyltransferase in complex in complex with CoA with and CoA a histoneand a H3histone peptide H3 (PDB peptide ID:1QSN (PDB [ID:1QSN70]); (D) the[70]); histone (D) the acetyltransferase histone acetyltransferase domain of hPCAF domain in of complex hPCAF with in CoAcomplex (PDB with ID: 1CM0CoA (PDB [73]); ID: and 1CM0 (E) yeast[73]); Hpa2and (E histone) yeast Hpa2 acetyltransferase histone acetyltransferase in complex with in complex AcCoA with (one dimerAcCoA of (one the tetramer) dimer of (PDBthe tetramer) ID: 1QSM (PDB [76]). ID: In 1QSM HAT1 [76]). and Esa1,In HAT1 the N-terminaland Esa1, the domain N-terminal is colored domain cyan, C-terminalis colored cyan, domain C-terminal is colored domain wheat, is andcolored conserved wheat, and conserved non-conserved and non-conserved motifs of thecentral motifs of GNAT the domaincentral GNAT are colored domain as in are Figure colored1. as in Figure 1.

Int. J. Mol. Sci. 2016, 17, 1018 11 of 45

2.3. Non-Histone Protein N-Acetyltransferase Family The GNAT superfamily includes many enzymes that acetylate non-histone proteins. Protein acetyltransferase from Sulfolobus solfataricus (SsPAT) [78], human α-tubulin acetyltransferase 1 (αTAT1) [79], human Naa50p [80] and M. tuberculosis AcCoA synthetase N-acetyltransferase (Rv0998) [81] are representative members of this family, the structures of which have been characterized. Non-histone protein acetylation plays an important role in regulation of mRNA and protein stability, subcellular localization and breakdown of proteins and modulation of protein-protein and DNA-non-histone protein interactions [82,83]. Acetylation of α-tubulin (on the ε-amino group of K40) by α-tubulin N-acetyltransferase 1 (αTAT1, EC 2.3.1.108) is involved in various microtubule-based processes in humans [79], mice [84] and other organisms, including Caenorhabditis elegans [85]. Structural analysis of 240-amino-acid human αTAT1 in complex with AcCoA showed that it has a “P-loop” Q131-R132-H133-G134-H135-G136 and a β-bulge involving L122 and D123 in the β4 strand [79]. The central β sheet is splayed apart to form a V-shaped AcCoA binding site (Figure5A). Interestingly, αTAT1 has an additional 12-residue β-hairpin structure between strands β4 and β5, which forms part of the α-tubulin binding site (Figure5A). Detailed analysis of the active site revealed that the conserved D157 and/or C120 residue could serve as a general base in the reaction. Mutagenesis studies confirmed the essential role of these two residues in catalysis [79]. An acetyl transfer mechanism involving two general base residues was also suggested for aralkylamine N-acetyltransferase (H120 and H122) [86] and Naa50p (H112 and Y173) [80]. The bisubstrate kinetics study showed that the reaction catalyzed by αTAT1 follows a ternary complex mechanism that does not involve a covalent protein/substrate intermediate [79]. The crystal structures of αTAT1 homologs from different species, including αTAT1 from mice [84] and Mec17 from human [87], were recently reported. In M. tuberculosis, fusion of a cyclic nucleotide binding domain with a GCN5-like catalytic PAT domain within Rv0998 (also known as MtPatA) enables direct cAMP control of protein acetylation, whereupon cAMP binding allosterically regulates the PAT activity [88]. Although the physiological substrate of MtPatA is yet to be identified, it was shown that its ortholog from M. smegmatis acetylates both M. smegmatis and M. tuberculosis acetyl-CoA synthetase in vitro [89]. MtPatA comprises three domains: an N-terminal cNMP-binding domain (residues 12–142), a central catalytic GNAT domain (residues 146–314) and a C-terminal extension (residues 315–333). MtPatA was crystallized as a monomer (Figure5B) [ 81]. Structural analysis of MtPatA in complex with AcCoA and cAMP revealed that cAMP binds at the N-terminal regulatory domain, AcCoA binds in the V-shaped cleft at the central catalytic domain, and the C-terminal extension serves as a regulatory element [81]. Kinetic and mutagenesis studies confirmed that E235 could serve as a general base, while R184 decreases the pKa of the ε-amino group of the Lys side chain of the substrate and thereby plays an important role in catalysis [81]. In contrast to other members of the non-histone protein N-acetyltransferase family, MtPatA can also efficiently catalyze transfer of alternative acyl groups including propionyl-CoA and butyryl-CoA. Protein lysine acetyltransferase of S. solfataricus (SsPAT, EC 2.3.2.-) catalyzes the acetylation of the K16 residue of the archaeal DNA-binding protein ALBA (acetylation lowers binding affinity) to lower its binding capacity as a means of chromatin regulation [78]. Structural analysis of SsPAT in complex with CoA showed that the cofactor molecule binds in the V-shaped cleft between strands β4 and β5 such that its diphosphate arm interacts with the enzyme’s “P-loop” [78]. As with 1 1 1 EcAAC(6 )-Ib, SeAAC(6 )-Ib11 and EfAAC(6 )-Ii, the β7 strand of SsPAT is placed between the β5 and β6 strands [21,25,30] (Figure5C). Interestingly, SsPAT has a unique structural feature (a “bent helix” comprised of residues 32–41) that might be involved in auto-regulation of the enzyme’s acetyltransferase activity. Detailed analysis of the active site revealed that there is no single residue that could solely serve as a general acid or base in the reaction catalyzed by SsPAT [78], although mutagenesis studies suggested that a set of well-ordered residues (Y38, E42, E43, D53, H72 and E76) might serve as a “proton wire” to shuttle a proton from the active site [78]. Int. J. Mol. Sci. 2016, 17, 1018 12 of 45

HumanInt. J. Mol. Sci.N( 2016α)-acetyltransferase, 17, 1018 Naa50p (hNaa50p, EC 2.3.2.-) transfers an acetyl group12 of onto 44 the α-aminohas a group β-bulge of in the strand N-terminal β4 (residues methionine M75 and T76) residue and ina typical proteins “P-loop” to regulate R84-R85-L86-G87-I88-G89 genome integrity [90]. It hasthat a β interacts-bulge in with strand the pyrophosphateβ4 (residues M75 arm andof CoA T76) [80]. and Structural a typical analysis “P-loop” of the R84-R85-L86-G87-I88-G89 complex of hNaa50p that interactswith CoA with and thea peptide pyrophosphate revealed that arm hNaa50p of CoA has [80 ].a Structuralunique 14-residue analysis β6– ofβ the7 hairpin complex that of forms hNaa50p withpart CoA of and the substrate-binding a peptide revealed site thatand determines hNaa50p hasthe aspecificity unique 14-residuefor the N-terminalβ6–β7 α hairpin-amino groups that forms part of(Figure the substrate-binding 5D). Mutagenesis, kineti site andc and determines structural studies the specificity suggested for that the Y73 N-terminal and H112 αcan-amino serve groupsas (Figurethe5 D).general Mutagenesis, base residues, kinetic deprotonating and structural the amin studieso group suggested of the thatsubstrate Y73 and via H112a water can molecule, serve as the generaland base that residues,at least one deprotonating of these residues the aminocan also group act as of a thegeneral substrate acid in via the a waterreaction molecule, catalyzedand by that hNaa50p [80]. at least one of these residues can also act as a general acid in the reaction catalyzed by hNaa50p [80].

FigureFigure 5. Cartoon5. Cartoon representation representation of of the the structures structures of non-histone of non-histone protein protein N-acetyltransferases.N-acetyltransferases. (A) (A) HumanHumanα α-tubulin-tubulin acetyltransferase in in complex complex with with AcCoA AcCoA (PDB (PDB ID: ID:4GS4 4GS4 [79]); [79 (B]);) M. (B )tuberculosisM. tuberculosis MtPat in complex with cAMP and AcCoA (PDB ID: 4AB [81]); (C) N-acetyltransferase from Sulfolobus MtPat in complex with cAMP and AcCoA (PDB ID: 4AB [81]); (C) N-acetyltransferase from solfataricus in complex with CoA (PDB ID: 3F8K [78]); and (D) Human N-acetyltransferase Naa50p in Sulfolobus solfataricus in complex with CoA (PDB ID: 3F8K [78]); and (D) Human N-acetyltransferase complex with CoA and peptide MLGPEGGRWGRPVGRRRRP (PDB ID: 3TFY [80]). The conserved Naa50p in complex with CoA and peptide MLGPEGGRWGRPVGRRRRP (PDB ID: 3TFY [80]). and non-conserved motifs of the GNAT domains, cofactors and substrates are colored as in Figure 3. The conserved and non-conserved motifs of the GNAT domains, cofactors and substrates are colored as2.4. in Arylalkylamine Figure3. N-Acetyltransferase Family (AANAT, EC 2.3.1.87) Arylalkylamine N-acetyltransferase (AANAT), also known as aralkylamine N-acetyltransferase 2.4. Arylalkylamine N-Acetyltransferase Family (AANAT, EC 2.3.1.87) or serotonin N-acetyltransferase, catalyzes transfer of an acetyl group from AcCoA to the primary Arylalkylamineamine of a wide rangeN-acetyltransferase of arylalkylamine (AANAT), substrates, including also known dopamine, as aralkylamine serotonin, Nphenylethylamine-acetyltransferase or serotoninand tryptamineN-acetyltransferase, [91]. It catalyzes catalyzes the penultimate transfer of step an in acetyl the biosynthesis group from of AcCoAmelatonin to from the primary serotonin, amine of a wideand thereby range ofcontrols arylalkylamine coherent coordination substrates, includingof the sleep-wake dopamine, cycle serotonin, in vertebrates phenylethylamine and seasonal and tryptaminereproduction [91]. It in catalyzes animals [91]. the penultimate step in the biosynthesis of melatonin from serotonin, and Ovis aries (sheep) AANAT (OaAANAT, 174 aa) exists as a monomer both in solution and in the thereby controls coherent coordination of the sleep-wake cycle in vertebrates and seasonal reproduction crystal [77,86]. Structural analysis of OaAANAT in complex with a bisubstrate analog, CoA-S- in animalsacetyltryptamine, [91]. revealed that it has a conserved “P-loop” R131-Q132-Q133-G134-K135-G136 and a Ovisβ-bulge aries at A123(sheep) and AANATH122 in the (OaAANAT, β5 strand. The 174 V-shaped aa) exists cavity as abetween monomer the β both4 and in β5 solution strands and in theaccommodates crystal [77, 86AcCoA]. Structural (Figure 6) analysis [77,86]. The of OaAANATlast β-strand in(β7) complex is placed withbetween a bisubstrate the β5 and β analog,6 CoA-strands.S-acetyltryptamine, This structural revealed feature thatis also it hasfound a conserved in some other “P-loop” GNAT R131-Q132-Q133-G134-K135-G136 enzymes [10]. The binding of and aAcCoAβ-bulge induces at A123 major and structural H122 in therearrangementsβ5 strand. Theat the V-shaped active site cavity of OaAANAT between that the βcompletes4 and β5 the strands accommodatesserotonin binding AcCoA pocket. (Figure This6)[ provided77,86]. Thea struct lasturalβ-strand basis for (β the7) isordered placed sequential between mechanism the β5 and β6 strands. This structural feature is also found in some other GNAT enzymes [10]. The binding of AcCoA induces major structural rearrangements at the active site of OaAANAT that completes the serotonin Int. J. Mol. Sci. 2016, 17, 1018 13 of 45 binding pocket. This provided a structural basis for the ordered sequential mechanism where the bindingInt. J. of Mol. AcCoA Sci. 2016 at, 17 the, 1018 active site of the enzyme is followed by the binding of the substrate tryptamine.13 of 44 The rate-limiting step in the acetylation reaction is the diffusional release of the product [92]. The Y168 where the binding of AcCoA at the active site of the enzyme is followed by the binding of the is positioned appropriately to serve as a general acid, and its function was confirmed by mutagenesis substrate tryptamine. The rate-limiting step in the acetylation reaction is the diffusional release of the studiesproduct [77]. [92]. Mutation The Y168 of theis positioned Y168 to Pheappropriately resulted into aserve ~30-fold as a general decrease acid, of and the itsV maxfunctionand awas 27-fold increaseconfirmed in the KbyM mutagenesisvalue of the studies enzyme [77]. [77 Mutation]. of the Y168 to Phe resulted in a ~30-fold decrease Structuresof the Vmax and of a the27-fold OaAANAT increase in the homologs KM value fromof the enzymeDrosophila [77]. melanogaster [93] and mosquito Aedes aegyptiStructures[94] have of the also OaAANAT been reported. homologs Mosquito from Drosophila AANATs melanogaster (aaNAT2, [93] aaNAT5b and mosquito and paaNAT7)Aedes haveaegypti some [94] unique have features:also been reported. the presence Mosquito of helix/helices AANATs (aaNAT2, between aaNAT5b the β and3 and paaNAT7)β4 strands, have and a differentsome unique active sitefeatures: residue the (Val/Ala) presence of corresponding helix/helices between to the conserved the β3 and Y168 β4 strands, residue and of OaAANAT a different [94]. Site-directedactive site mutagenesis, residue (Val/Ala) kinetic corresponding studies and to pH-ratethe conserved profiling Y168 of residueD. melanogaster of OaAANATAANAT [94]. Site- enzyme (DmAANAT)directed mutagenesis, confirmed thatkinetic it followsstudies anand ordered pH-rate sequentialprofiling of Bi-Bi D. melanogaster reaction mechanism AANAT enzyme where E47 (DmAANAT) confirmed that it follows an ordered sequential Bi-Bi reaction mechanism where E47 serves as a remote general base that extracts a proton from the positively charged amino group of the serves as a remote general base that extracts a proton from the positively charged amino group of the acceptoracceptor substrate substrate via avia “proton a “proton wire” wire” [93 [93,95].,95]. The The substitution substitution E47AE47A resulted in in a a~15-fold ~15-fold decrease decrease of the kcat,appof the kvaluecat,app value compared compared to wild-type to wild-type [95 [95].].

FigureFigure 6. Cartoon 6. Cartoon representation representation of of the the structure structure of of arylalkylamine arylalkylamine N-acetyltransferase-acetyltransferase of ofOvisOvis aries aries in complexin complex with a with bisubstrate a bisubstrate analog, analog, CoA- CoA-S-acetyltryptamineS-acetyltryptamine (PDB (PDB ID: ID: 1CJW 1CJW [ 77[77]).]). TheThe conservedconserved and and non-conserved motifs of the GNAT domain are colored as in Figure 3. non-conserved motifs of the GNAT domain are colored as in Figure3. 2.5. Glucosamine-6-Phosphate N-Acetyltransferase 1 Family (GNA1, EC 2.3.1.4) 2.5. Glucosamine-6-Phosphate N-Acetyltransferase 1 Family (GNA1, EC 2.3.1.4) Glucosamine-6-phosphate N-acetyltransferase 1 (GNA1) catalyzes transfer of an acetyl group Glucosamine-6-phosphatefrom AcCoA to the primaryN -acetyltransferaseamine of D-glucosamine-6-phosphate 1 (GNA1) catalyzes (GlcN6P) transfer to ofform an N acetyl- groupacetylglucosamine-6-phosphate from AcCoA to the primary (GlcNAc-6P) amine [11]. of TheD-glucosamine-6-phosphate biosynthesis of GlcNAc-6P is a (GlcN6P) key step in tothe form N-acetylglucosamine-6-phosphateformation of the energy-rich metabolite (GlcNAc-6P) UDP-N [-acetyl-glucosamine11]. The biosynthesis (UDP-GlcNAc) of GlcNAc-6P [11,96]. is aUDP- key step in theGlcNAc formation serves of as the a precursor energy-rich in the metabolitehexosamine UDP-biosynthesisN-acetyl-glucosamine pathway that is linked (UDP-GlcNAc) to the synthesis [11 ,96]. UDP-GlcNAcof major metabolites serves as aof precursorglycolysis, inlipid the synthesis, hexosamine tricarboxylic biosynthesis acid cycle pathway and nitrogen that is linkedcycle. In to the vertebrates, UDP-GlcNAc also acts as a precursor to generate UDP-N-acetylgalactosamine and synthesis of major metabolites of glycolysis, lipid synthesis, tricarboxylic acid cycle and nitrogen cytosine monophosphate (CMP)-N-acetylneuraminic acid. In addition, UDP-GlcNAc serves as a cycle.substrate In vertebrates, for chitin UDP-GlcNAc synthase and also phosphatidylinositol- acts as a precursorN to-acetylglucosaminyltransferase generate UDP-N-acetylgalactosamine (which and cytosinecatalyzes the monophosphate first step in the (CMP)-biosynthesisN-acetylneuraminic pathway for glycophosphatidylinositol acid. In addition, UDP-GlcNAc (GPI) anchors of serves as a substratevarious proteins) for chitin [96–98]. synthase and phosphatidylinositol-N-acetylglucosaminyltransferase (which catalyzesStructural the first stepand inbiochemical the biosynthesis analysis pathwayof S. cerevisiae for glycophosphatidylinositol GNA1 (ScGNA1, 161 aa) (GPI)showed anchors that of variousScGNA1 proteins) exists [ 96as– a98 dimer]. both in solution and in the crystal (Figure 7) [99]. The two parallel β-strands Structural(β4 and β5) and of biochemicalScGNA1 are analysis splayed of apartS. cerevisiae to generateGNA1 the (ScGNA1, characteristic 161 aa)V-shaped showed cleft that that ScGNA1 existsaccommodates as a dimer both AcCoA. in solution ScGNA1 and has in an the atypical crystal “P-loop” (Figure 7G108-Q109-G110-L11-G112-K113)[ 99]. The two parallel β-strands that (β4 interacts with the diphosphate arm of AcCoA. It also has a β-bulge structure in strand β4 formed by and β5) of ScGNA1 are splayed apart to generate the characteristic V-shaped cleft that accommodates residues D99 and I100 [99]. The β6 strand of each monomer is projected away from the core fold and AcCoA. ScGNA1 has an atypical “P-loop” G108-Q109-G110-L11-G112-K113 that interacts with the forms part of the β-sheet of the opposite monomer, so that the two halves of the dimer are stabilized β β diphosphatethrough “strand arm of swapping”. AcCoA. It C-terminal also has a strand-bulge swa structurepping was in also strand observed4 formed in the structures by residues of D99 and I100 [99]. The β6 strand of each monomer is projected away from the core fold and forms

Int. J. Mol. Sci. 2016, 17, 1018 14 of 45 part of the β-sheet of the opposite monomer, so that the two halves of the dimer are stabilized through “strand swapping”. C-terminal strand swapping was also observed in the structures of Int. J. Mol. Sci. 2016, 17, 1018 14 of 44 ScHpa2 and SeAAC(61)-Iy [10,76]. Detailed structural analysis revealed that the binding site for the acceptorScHpa2 substrate and SeAAC(6 is located′)-Iy at[10,76]. the dimer Detailed interface. structural The analysis hydroxyl revealed group that of Y143the binding is within site afor hydrogen the bondingacceptor distance substrate from is located the sulfur at the atomdimer ofinterface. AcCoA, The suggesting hydroxyl group that of Y143 Y143 mayis within serve a hydrogen as a general acidbonding and donate distance a proton from the to sulfur the leaving atom of thiolateAcCoA, suggesting anion. Mutagenesis that Y143 may data serve supported as a general the acid crucial role ofand Y143 donate for a theproton activity to the of leaving the ScGNA1 thiolate anion. enzyme Mutagenesis [11]. No data candidates supported for the a generalcrucial role base of were identifiedY143 for in the the activity ScGNA1 of the structure. ScGNA1 enzyme However, [11]. the No acceptorcandidates substrate for a general in thebase reaction were identified catalyzed in by the ScGNA1 structure. However, the acceptor substrate in the reaction catalyzed by ScGNA1 ScGNA1 (GlcN6P) has a much lower pKa (7.75) than that of other GNAT acceptor substrates and is (GlcN6P) has a much lower pKa (7.75) than that of other GNAT acceptor substrates and is likely to likely to bind to the enzyme in a deprotonated form, eliminating the need for a general base [99]. bind to the enzyme in a deprotonated form, eliminating the need for a general base [99]. The The structuresstructures of of the the ScGNA1 ScGNA1 homologs homologs from from HomoHomo sapiens sapiens [100,101],[100,101 Aspergillus], Aspergillus fumigatus fumigatus [100],[ 100], ArabidopsisArabidopsis thaliana thaliana[102 [102]] and andC. C. elegans elegans[ 103[103]] werewere also reported. reported. The The main-chain main-chain oxygen oxygen of E156 of E156of of humanhuman GNA1 GNA1 (hGNA1) (hGNA1) forms forms a hydrogen a hydrogen bond bond with with the the GlcN6P’s GlcN6P’s amino group group and and thus thus increases increases its nucleophilicits nucleophilic nature. nature. The essential The essential role ofrole E156 of E156 in the in the acceptor acceptor substrate substrate binding binding and and inin catalysiscatalysis was confirmedwas confirmed by mutagenesis by mutagenesis [101]. [101].

FigureFigure 7. 7.Cartoon Cartoonrepresentation representation of of the the structure structure of dimeric of dimeric yeast yeastglucosamine-6-phosphate glucosamine-6-phosphate N- acetyltransferase 1 in complex with CoA and N-acetyl-D-glucosamine-6-phosphate (GlcNAc-6P) (PDB N-acetyltransferase 1 in complex with CoA and N-acetyl-D-glucosamine-6-phosphate (GlcNAc-6P) ID: 1I1D [99]). The conserved and non-conserved motifs of the GNAT domain, cofactor and substrates (PDB ID: 1I1D [99]). The conserved and non-conserved motifs of the GNAT domain, cofactor and are colored as in Figure 3. substrates are colored as in Figure3. 2.6. Microcin C7 Self-Immunity Acetyltransferase Family (MccE) 2.6. Microcin C7 Self-Immunity Acetyltransferase Family (MccE) Microcin C7 (McC) is a highly potent antibiotic, produced by certain E. coli strains, that can Microcininhibit the C7growth (McC) of isenteric a highly bacteria potent by blocki antibiotic,ng aspartyl-tRNA produced syntheta by certainse andE. colithusstrains, halting thatthe can inhibitcellular the growth protein translation of enteric mechanism bacteria by [104]. blocking It is composed aspartyl-tRNA of heptapeptide synthetase MRTGNAD and thus conjugated halting the cellularto modified protein translationAMP. The E. mechanism coli MccE protein [104]. acetylates It is composed McC converting of heptapeptide it into a form MRTGNAD that is non-toxic conjugated to the cell, thus conferring E. coli resistance to this compound [104,105]. to modified AMP. The E. coli MccE protein acetylates McC converting it into a form that is non-toxic to The acetyltransferase domain of MccE (188 aa) exists as a monomer both in solution and in the the cell, thus conferring E. coli resistance to this compound [104,105]. crystal [104]. The β4 and β5 strands are splayed apart to form the AcCoA binding site, and the last β7 Theis placed acetyltransferase between the β domain5 and β of6 strands MccE (188(Figure aa) 8). exists Unlike as aother monomer GNAT bothproteins, in solution MccE has and in the crystalan additional [104]. The70 aminoβ4 and acidβ 5residues strands at are its N-terminus splayed apart that toform form part the of AcCoAthe substrate binding binding site, site and the last β(Figure7 is placed 8). Structural between analysis the β5 of and MccEβ6 in strands complex (Figure with AcCoA8). Unlike and othersynthetic GNAT sulfamoyl proteins, adenylate MccE has an additionalsubstrates 70(aspartyl-sulfamoyl amino acid residues adenosine at its and N-terminus glutamyl-sulfamoyl that form adenosine) part of therevealed substrate that T453 binding and site (FigureF4668). form Structural π-stacking analysis interactions of MccE with in the complex adenine with ring AcCoA of the substrate and synthetic analog, sulfamoyland thus play adenylate an substratesimportant (aspartyl-sulfamoyl role in the substrate adenosine specificity and[104]. glutamyl-sulfamoyl The carbonyl oxygen adenosine)of AcCoA interacts revealed with that the T453 and F466main-chain form πamide-stacking group interactions of Y510 (~3.1 with Å) and the backbone adenine ringcarbonyl of the of I508. substrate Residues analog, S553 andand thusE572 play were proposed to serve as a general acid and a general base in the MccE-catalyzed reaction, an important role in the substrate specificity [104]. The carbonyl oxygen of AcCoA interacts with respectively. An S553A/E572A variant of MccE showed significantly reduced enzymatic activity the main-chain amide group of Y510 (~3.1 Å) and backbone carbonyl of I508. Residues S553 and (~25-fold), confirming their importance for catalysis [105]. Although the side chain of C546 was found

Int. J. Mol. Sci. 2016, 17, 1018 15 of 45

E572 were proposed to serve as a general acid and a general base in the MccE-catalyzed reaction, respectively. An S553A/E572A variant of MccE showed significantly reduced enzymatic activity Int. J. Mol. Sci. 2016, 17, 1018 15 of 44 (~25-fold), confirming their importance for catalysis [105]. Although the side chain of C546 was found disulfide-bondeddisulfide-bonded to the to the CoA CoA thiol thiol in in the the crystal, crystal, mutagenesis mutagenesis data data on on MccE MccE homologs homologs and and the lack the lackof of conservationconservation of this of this residue residue rule rule out out the the possibility possibility thatthat MccE catalyzes catalyzes acetyl acetyl transfer transfer by a by ping-pong a ping-pong mechanismmechanism [104 ].[104].

FigureFigure 8. Cartoon 8. Cartoon representation representation of of the the structure structure ofof thethe acetyltransferase domain domain of E. of coliE. coliMccEMccE in in complex with AcCoA and adenosine monophosphate (AMP) (PDB ID: 3R96 [104]). The conserved complex with AcCoA and adenosine monophosphate (AMP) (PDB ID: 3R96 [104]). The conserved and and non-conserved motifs of the GNAT domain, cofactor and substrate are colored as in Figure 3. non-conserved motifs of the GNAT domain, cofactor and substrate are colored as in Figure3. 2.7. Pseudaminic Acid Biosynthesis Protein H Family (PseH, EC 2.3.1.202) 2.7. Pseudaminic Acid Biosynthesis Protein H Family (PseH, EC 2.3.1.202) Pseudaminic acid biosynthesis protein H (PseH) catalyzes the third step of the biosynthesis Pseudaminicpathway for pseudaminic acid biosynthesis acid in proteinHelicobacter H (PseH) pylori and catalyzes Campylobacter the third jejuni step toof form the UDP-2,4- biosynthesis pathwaydiacetamido-2,4,6-trideoxy- for pseudaminic acidβ-L-altropyranose in Helicobacter (UDP-sugar) pylori and Campylobacterfrom UDP-4-amino-4,6-dideoxy- jejuni to form UDP-2,4-β-L- diacetamido-2,4,6-trideoxy-AltNAc using AcCoA asβ an-L-altropyranose acetyl donor [106]. (UDP-sugar) PseH plays from an important UDP-4-amino-4,6-dideoxy- role in flagella assemblyβ-L -AltNAcand usingfunction. AcCoA asMutation an acetyl of the donor pseH [ 106gene]. in PseH C. jejuni plays resulted an important in non-moti rolele in phenotype flagella assembly [106]. and function. H. pylori PseH (HpPseH, molecular weight (MW) 21.1 kDa) exists as a homodimer in solution, Mutation of the pseH gene in C. jejuni resulted in non-motile phenotype [106]. although there are three subunits of PseH in the crystal [107]. Two of these subunits form a non- H. pylori PseH (HpPseH, molecular weight (M ) 21.1 kDa) exists as a homodimer in solution, crystallographic dimer similar to that found in the Wcrystals of S. typhimurium RimL [108], while the althoughthird theresubunit are forms three a similar subunits dimerof with PseH a symmetry- in therelated crystal subunit. [107]. Structur Two ofal analysis these subunitsof HpPseH form a non-crystallographicin complex with AcCoA dimer showed similar that to the that β4 foundand β5 instrands the crystalsare splayed of S.apart, typhimurium creating a RimLchannel [ 108], whilethrough the third the subunit molecule forms that accommodates a similar dimer AcCoA with (Figure a symmetry-related 9). The β7 strand subunit. is sandwiched Structural between analysis of HpPseHthe β5 inand complex β6 strands. with It AcCoAhas an atypical showed “P-loop” that the thatβ4 andinteractsβ5 strands with the are pyrophosphate splayed apart, arm creating of a channelAcCoA. through The β-bulge the molecule structure that is missing accommodates in HpPseH. AcCoA Furthermore, (Figure it9 ).has The an βadditional7 strand β is-strand sandwiched at betweenthe N-terminus the β5 and andβ6 strands.an additional It has α-helix an atypical at the C-terminal “P-loop”that end.interacts The putative with catalytic the pyrophosphate site is located arm of AcCoA.within The eachβ monomer.-bulge structure At the dimer is missing interface, in HpPseH. the C-terminal Furthermore, β-strands it from has aneach additional monomer βform-strand a at continuous β-sheet similar to that present in EfAAC(6′)-Ii [34] and ScGNA1 [99]. The carbonyl oxygen the N-terminus and an additional α-helix at the C-terminal end. The putative catalytic site is located of AcCoA is hydrogen-bonded to the main-chain amide of I93 and the OH group of Y138. Detailed β withinanalysis each monomer. of the modeled At theternary dimer complex interface, (HpPseH/ the C-terminalAcCoA/UDP-sugar)-strands showed from that each the monomer side chain form 1 a continuousof the conservedβ-sheet S78 similar residue to could that act present as a remote in EfAAC(6 general base)-Ii [to34 extract] and ScGNA1a proton from [99]. the The 4-amino carbonyl oxygengroup of AcCoAof UDP-sugar is hydrogen-bonded via a well-ordered to water the main-chainmolecule. The amide structure of also I93 andsuggested the OH that group conserved of Y138. DetailedY138 analysis could serve of the as a modeled general acid ternary in catalysis, complex consistent (HpPseH/AcCoA/UDP-sugar) with the role of the corresponding showed residue that the side chainin other of GNAT the conserved proteins [107]. S78 residue HpPseH could and its act homolog as a remote from C. general jejuni (CjPseH) base to extractshare many a proton structural from the 4-aminofeatures group [109], of UDP-sugaralthough in contrast via a well-ordered to HpPseH, CjPseH water molecule.exists as a monomer The structure in solution also suggested and in the that conservedcrystal Y138 [109]. could serve as a general acid in catalysis, consistent with the role of the corresponding residue in other GNAT proteins [107]. HpPseH and its homolog from C. jejuni (CjPseH) share many

Int. J. Mol. Sci. 2016, 17, 1018 16 of 45 structural features [109], although in contrast to HpPseH, CjPseH exists as a monomer in solution and inInt. the J. Mol. crystal Sci. 2016 [109, 17]., 1018 16 of 44

Figure 9. Cartoon representation of the structure of Helicobacter pylori pseudaminic acid biosynthesis protein H in complex with AcCoA (PDB ID: 4RI1 [107107]).]). The conserved and non-conserved motifs of the GNAT domaindomain areare coloredcolored asas inin FigureFigure3 3..

2.8. Thymidine Diphosphate (TDP)-Fucosamine Acetyltransferase Family (WecD, ECEC 2.3.1.210)2.3.1.210) E. colicoliTDP-fucosamine TDP-fucosamine acetyltransferase acetyltransferase (WecD, (WecD, 235 aa) 235 catalyzes aa) catalyzes the final stepthe infinal the biosynthesisstep in the ofbiosynthesis an outer membraneof an outer glycolipidmembrane termed glycolipid enterobacterial termed enterobacterial common antigen common (ECA). antigen It acetylates(ECA). It acetylates dTDP-4-amino-4,6-dideoxy-α-D-galactose to TDP-4-acetamido-4,6-dideoxy-D-galactose dTDP-4-amino-4,6-dideoxy-α-D-galactose to TDP-4-acetamido-4,6-dideoxy-D-galactose using AcCoA asusing an acetylAcCoA donor. as an ECAacetyl is donor. widely ECA distributed is widely among distributed Enterobacteriaceae among Enterobacteriaceae and plays an important and plays role an inimportant bacterial role resistance in bacterial to organic resistance acids andto organic intestinal acids bile and salts intestinal used as abile defense salts mechanismused as a defense by the eukaryoticmechanism host by the [110 eukaryotic,111]. It also host plays [110,111]. a crucial It rolealso inplays flagella a crucial biosynthesis, role in flagella and thus biosynthesis, affects bacterial and motilitythus affects and bacterial virulence motility [112]. and virulence [112]. WecD existsexists asas aa dimerdimer bothboth inin solutionsolution andand inin thethe crystalcrystal [[113].113]. Structural analysis of the WecD/AcCoA binary binary complex complex revealed revealed that that each monomer harbors a typical C-terminal GNAT domain (residues(residues 7070 toto 218) 218) and and an an N-terminal N-terminal partial partial GNAT GNAT domain domain (residues (residues 3 to 3 69, to and69, and 219 to219 224) to lacking224) lacking the first the two firstα two-helices α-helices and two andβ -strandstwo β-strands (Figure (Figure 10). The 10). C-terminal The C-terminal domain domain contains contains a β-bulge a involvingβ-bulge involving G66 and G66 L167 and of the L167β8 of strand, the β and8 strand, an atypical and an “P-loop” atypical A169-G170-R171-G172-A173-G174. “P-loop” A169-G170-R171-G172- AcCoAA173-G174. binds AcCoA in the structurallybinds in the conserved structurally V-shaped conserved cleft atV-shaped the C-terminal cleft at GNAT the C-terminal domain. Detailed GNAT analysisdomain. ofDetailed the modeled analysis ternary of the complex modeled WecD/AcCoA/TDP-fucosamine ternary complex WecD/AcCoA/TDP-fucosamine showed that the conserved showed Y208that the residue conserved would Y208 be within residue the would hydrogen be within bonding the distancehydrogen (~3.0 bonding Å) of distance the thioester (~3.0 moietyÅ) of the of AcCoA,thioester and moiety that theof AcCoA, backbone and carbonyl that the oxygen backbone of A196 carbonyl could formoxygen hydrogen of A196 bonds could with form the hydrogen 4-amino groupbonds ofwith the the substrate. 4-amino Like group in mostof the other substrate. GNAT Like enzymes, in most Y208 other is believedGNAT enzymes, to play an Y208 important is believed role into theplay reaction an important by (i) ensuring role in the the reaction proper positioningby (i) ensuring of the the acetyl proper group positioning for transfer; of the and acetyl (ii) stabilizing group for andtransfer; protonating and (ii) stabilizing the thiolate and anion protonating of the leaving the thio CoAlate [113 anion]. Mutation of the leaving of the correspondingCoA [113]. Mutation tyrosine of residuethe corresponding in ScGNA1 andtyrosine serotonin residueN-acetyltransferase in ScGNA1 and confirmed serotonin its N essential-acetyltransferase role in catalysis confirmed [11,77, 86its]. Althoughessential role no residuein catalysis in the[11,77,86]. immediate Although vicinity no of residue the substrate in the immediate was identified vicinity as aof likely the substrate general base,was identified the structural as a likely analysis general suggested base, thatthe structur E68, whichal analysis was ~7.5 suggested Å away that from E68, the which 4-amino was group ~7.5 inÅ theaway modeled from the Michaelis 4-amino complex,group in couldthe modeled extract aMichaelis proton from complex, the amino could groupextract of a theproton substrate from viathe wateramino molecules. group of the substrate via water molecules.

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Int. J. Mol. Sci. 2016, 17, 1018 17 of 44

Figure 10. Cartoon representation of the structure of E. coli WecD in complex with AcCoA. The N- Figure 10. Cartoon representation of the structure of E. coli WecD in complex with AcCoA. terminal partial GNAT domain is colored cyan (PDB ID: 2FT0 [113]). The conserved and non- The N-terminal partial GNAT domain is colored cyan (PDB ID: 2FT0 [113]). The conserved and conserved motifs of the GNAT domain are colored as in Figure 3. non-conserved motifs of the GNAT domain are colored as in Figure3. 2.9. TabtoxinFigure 10. Resistance Cartoon representationProtein Family of (TTR, the structure EC 2.3.1.-) of E. coli WecD in complex with AcCoA. The N- 2.9. Tabtoxinterminal Resistance partial ProteinGNAT domain Family is (TTR, colored EC cyan 2.3.1.-) (PDB ID: 2FT0 [113]). The conserved and non- conservedTabtoxin resistancemotifs of the protein GNAT (TTR,domain M areW ~ colored19 kDa) as inactivates in Figure 3. tabtoxin (the progenitor of the highly Tabtoxinvirulent non-specific resistance bacterial protein toxin (TTR, tabtoxinineMW ~19 β-lactam kDa) inactivates (TβL)) by acetylation, tabtoxin and (the thus progenitor gives self- of the highly2.9.immunity virulent Tabtoxin to non-specific Resistance tabtoxin-producing Protein bacterial Family pathogens, toxin (TTR, tabtoxinine EC such 2.3.1.-) as P.β -lactamsyringae (T[114,115].βL)) by T acetylation,βL is the predominant and thus gives phytotoxin that causes the tobacco wildfire disease [114,115]. In addition, TβL can kill microbes and self-immunityTabtoxin to tabtoxin-producingresistance protein (TTR, pathogens, MW ~ 19 kDa) such inactivates as P. syringae tabtoxin[114 (the,115 progenitor]. TβL is of the the predominant highly damage mammalian and plant cells by inhibiting glutamine synthetase that contributes to the phytotoxinvirulent thatnon-specific causes bacterial the tobacco toxin wildfire tabtoxinine disease β-lactam [114 (T,115βL))]. by In acetylation, addition, and TβL thus can gives kill microbesself- neutralization of cellular ammonia [116]. Transgenic plants that produce TTR can prevent wildfire and damageimmunity mammalian to tabtoxin-producing and plant pathogens, cells by inhibiting such as P. glutaminesyringae [114,115]. synthetase TβL is that the contributespredominant to the disease caused by P. syringae pv. Tabaci [117,118]. phytotoxin that causes the tobacco wildfire disease [114,115]. In addition, TβL can kill microbes and neutralizationStructural of cellular analysis ammonia of TTR in complex [116]. Transgenic with co-purified plants AcCoA that produce showed TTRthat this can protein prevent exists wildfire damage mammalian and plant cells by inhibiting glutamine synthetase that contributes to the diseaseas a caused dimer in by theP. syringaecrystal [114].pv. TabaciLike mo[117st other,118]. GNAT family members, TTR has a β-bulge, formed neutralization of cellular ammonia [116]. Transgenic plants that produce TTR can prevent wildfire Structuralby residues analysisQ94 and K95 of TTR on the in β complex4 strand, and with a co-purifiedcharacteristic AcCoA V-shaped showed AcCoA that binding this site protein between exists as disease caused by P. syringae pv. Tabaci [117,118]. a dimerstrands in the β4 crystaland β5 (Figure [114]. Like 11). It most harbors other the GNAT conserved family six-residue members, sequence TTR has(Arg/Gln)-X-X-Gly-X- a β-bulge, formed by Structural analysis of TTR in complex with co-purified AcCoA showed that this protein exists (Gly/Ala) in motif A that is responsible for the AcCoA recognition and binding. The binding mode of residuesas a dimer Q94 and in the K95 crystal on the [114].β4 Like strand, most and other a characteristic GNAT family V-shapedmembers, TTR AcCoA has a binding β-bulge, siteformed between AcCoA is similar to most other GNAT enzymes [10,76]. The carbonyl group of AcCoA forms a strandsby residuesβ4 and Q94β5 and (Figure K95 on11 ).the It β4 harbors strand, and the a conserved characteristic six-residue V-shaped AcCoA sequence binding (Arg/Gln)-X-X-Gly- site between hydrogen bond with the hydroxyl group of the conserved residue Y141. In the modeled ternary X-(Gly/Ala)strands β in4 and motif β5 A(Figure that is 11). responsible It harbors forthe theconserved AcCoA six-residue recognition sequence and binding. (Arg/Gln)-X-X-Gly-X- The binding mode complex TTR/AcCoA/TβL, the imide nitrogen of TβL is positioned ~4.7 Å from the carbonyl carbon of AcCoA(Gly/Ala) is similarin motif A to that most is responsible other GNAT for the enzymes AcCoA [recognition10,76]. The and carbonyl binding. The group binding of AcCoA mode of forms of AcCoA, while the imide carbon of TβL interacts with E92 and D130 via a well-ordered water a hydrogenAcCoA is bond similar with to themost hydroxyl other GNAT group enzymes of the conserved[10,76]. The residuecarbonyl Y141. groupIn of theAcCoA modeled formsternary a molecule (4.6 Å). This water molecule might be crucial for the initial deprotonation and thus a water- hydrogen bond with βthe hydroxyl group of the conservedβ residue Y141. In the modeled ternary complexmediated TTR/AcCoA/T direct acetyl L,transfer the imide mechanism nitrogen was of TproposedL is positioned for the TTR-mediated ~4.7 Å from thereaction. carbonyl E92 carbonand of complex TTR/AcCoA/TβL, the imide nitrogen of TβL is positioned ~4.7 Å from the carbonyl carbon AcCoA,D130 while are thought the imide to act carbon together of T asβL a interactsgeneral base with in E92catalysis, and D130 whilst via Y141 a well-ordered could serve as water a general molecule of AcCoA, while the imide carbon of TβL interacts with E92 and D130 via a well-ordered water (4.6 Å).acid This that waterdonates molecule a proton mightto the sulfur be crucial atom forof CoA the initial[114]. deprotonation and thus a water-mediated molecule (4.6 Å). This water molecule might be crucial for the initial deprotonation and thus a water- direct acetyl transfer mechanism was proposed for the TTR-mediated reaction. E92 and D130 are mediated direct acetyl transfer mechanism was proposed for the TTR-mediated reaction. E92 and thoughtD130 to are act thought together to act as atogether general as basea general in catalysis, base in catalysis, whilst Y141 whilst could Y141 servecould serve as a general as a general acid that donatesacid athat proton donates tothe a proton sulfur to atom the sulfur of CoA atom [114 of ].CoA [114].

Figure 11. Cartoon representation of the structure of the tabtoxin resistance protein from Pseudomonas syringae in complex with AcCoA (PDB ID: 1GHE [114]). The conserved and non-conserved motifs of the GNAT domain are colored as in Figure 3.

FigureFigure 11. 11. Cartoon representation representation of the of structure the structure of the tabtoxin of the resistance tabtoxin protein resistance from Pseudomonas protein from syringae in complex with AcCoA (PDB ID: 1GHE [114]). The conserved and non-conserved motifs of Pseudomonas syringae in complex with AcCoA (PDB ID: 1GHE [114]). The conserved and non-conserved the GNAT domain are colored as in Figure 3. motifs of the GNAT domain are colored as in Figure3.

Int. J. Mol. Sci. 2016, 17, 1018 18 of 45

2.10.Int. Mpr1 J. Mol. Family Sci. 2016 (EC, 17, 1018 3.4.1.-) 18 of 44 Mpr1, a 229-residue antioxidant enzyme, encoded by the sigma 1278b gene for proline-analog 2.10. Mpr1 Family (EC 3.4.1.-) resistance of S. cerevisiae, was initially reported as N-acetyltransferase that detoxifies the proline analogMpr1, a L229-residue-azetidine-2-carboxylate antioxidant enzyme, (AZC) encoded by by transforming the sigma 1278b it intogene forN-acetyl-AZC proline-analog [119]. resistance of S. cerevisiae, was initially reported as N-acetyltransferase that detoxifies the proline In addition, Mpr1 is involved in L-proline metabolism by acetylating intermediate molecules, analog L-azetidine-2-carboxylate (AZC) by transforming it into N-acetyl-AZC [119]. In addition, including L-∆1-pyrroline-5-carboxylate (P5C) and L-glutamate-γ-semialdehyde (GSA), and thus Mpr1 is involved in L-proline metabolism by acetylating intermediate molecules, including L-Δ1- plays an important role in defending yeast cells from oxidative stress [120–122]. While most GNAT pyrroline-5-carboxylate (P5C) and L-glutamate-γ-semialdehyde (GSA), and thus plays an important enzymesrole in acetylate defending primary yeast cells amines, from Mpr1oxidative acetylates stress [120–122]. only cyclic While secondary most GNAT amines enzymes such acetylate as AZC and cis-4-hydroxy-primary amines,L-proline Mpr1 (CHOP) acetylates [119 only]. cyclic secondary amines such as AZC and cis-4-hydroxy-L- Mpr1proline exists (CHOP) as [119]. a dimer in solution [119]. Analysis of the crystal structure of Mpr1 in complex with its naturalMpr1 exists substrate as a dimer CHOP in solution revealed [119]. that Analysis the β-bulge of the is crystal missing structure (Figure of Mpr112). Inin thecomplex modeled ternarywith complex its natural Mpr1/CHOP/AcCoA, substrate CHOP revealed the that sulfur the atomβ-bulge of is AcCoA missing interacts (Figure 12). with In thethe sidemodeled chain of ternary complex Mpr1/CHOP/AcCoA, the sulfur atom of AcCoA interacts with the side chain of N178. Substitution of N178 with alanine decreased the kcat ~40-fold, indicating that N178 is essential for catalysisN178. Substitution [119]. The of initialN178 with velocity alanine pattern decreased analysis the kcat revealed ~40-fold, indicating that the Mpr1-catalyzed that N178 is essential reaction for catalysis [119]. The initial velocity pattern analysis revealed that the Mpr1-catalyzed reaction proceeds via a direct acetyl transfer mechanism that involves formation of a tetrahedral intermediate. proceeds via a direct acetyl transfer mechanism that involves formation of a tetrahedral intermediate. The breakdown of this intermediate generates a thiolate anion which is stabilized by the interaction The breakdown of this intermediate generates a thiolate anion which is stabilized by the interaction withwith the side-chainthe side-chain amide amide of of N178, N178, and and then then aa waterwater molecule molecule bound bound to toN178 N178 donates donates a proton a proton to to releaserelease CoA. CoA. The useThe ofuse a side-chainof a side-chain amide amide of an of Asn an Asn residue residue in enzymatic in enzymatic catalysis catalysis is ais unique a unique feature of Mpr1.feature of Mpr1.

Figure 12. Cartoon representation of the structure of yeast Mpr1 in complex with cis-4-hydroxy-L- Figure 12. Cartoon representation of the structure of yeast Mpr1 in complex with cis-4-hydroxy- proline (CHOP) (PDB ID: 3W6X [119]). The conserved and non-conserved motifs of the GNAT L-proline (CHOP) (PDB ID: 3W6X [119]). The conserved and non-conserved motifs of the GNAT domain are colored as in Figure 3. domain are colored as in Figure3. 2.11. Spermidine/Spermine N1-Acetyltransferase Family (SSAT, EC 2.3.1.57) 2.11. Spermidine/Spermine N1-Acetyltransferase Family (SSAT, EC 2.3.1.57) Spermidine/spermine N1-acetyltransferase (SSAT) is one of the enzymes in the polyamine Spermidine/sperminedegradation pathway [123].N1-acetyltransferase It uses AcCoA (SSAT) as the is onedonor of thesubstrate. enzymes Acetylation in the polyamine of degradationspermidine/spermine pathway [123 promotes]. It uses AcCoAexpulsion as or the breakd donorown substrate. of polyamines. Acetylation Abnormally of spermidine/spermine high cellular promotespolyamine expulsion levels or in breakdown human cells ofare polyamines. associated with Abnormally different diseases, high cellular including polyamine Alzheimer’s, levels cystic in human fibrosis and cancer [123–125]. cells are associated with different diseases, including Alzheimer’s, cystic fibrosis and cancer [123–125]. Bacillus subtilis PaiA (BsPaiA, 172 aa) is a member of the SSAT family that exists as a monomer Bacillus subtilis PaiA (BsPaiA, 172 aa) is a member of the SSAT family that exists as a monomer in in solution and a dimer in the crystal [126]. The structure of BsPaiA has the characteristic V-shaped solutiongroove and for a dimer AcCoA in binding the crystal (Figure [126 ].13). The Interestingly, structure of the BsPaiA structural has the elements characteristic that are V-shaped likely to be groove for AcCoAinvolved binding in substrate (Figure binding 13). Interestingly, (the loop connecting the structural strands elements β3 and β that4 and are the likely C-terminal to be involvedregion in substratecomprising binding the (the β6 and loop β7 connecting strands) show strands significantβ3 and differenceβ4 ands thewith C-terminal other GNAT region proteins, comprising and are the β6 andthereforeβ7 strands) thought show to be significant important determinants differences with of the other substrate GNAT specificity proteins, of BsPaiA. and are One therefore of the two thought to besubunits important in the determinants asymmetric unit of the of the substrate crystal of specificity the BsPaiA/CoA of BsPaiA. binary One complex of the contained two subunits a single in the asymmetricCoA molecule, unit of and the the crystal other of an the oxidized BsPaiA/CoA CoA dimer. binary One complex of the monomers contained of athis single dimer CoA occupies molecule, and thethe othernatural an AcCoA oxidized binding CoA site, dimer. whereas One ofthe the second monomers CoA monomer of this dimeris bound occupies in the other thenatural end of the AcCoA active-site tunnel, which is where the acceptor substrate is thought to bind. Structural analysis of binding site, whereas the second CoA monomer is bound in the other end of the active-site tunnel, BsPaiA showed that the side chain of Y142, that interacts with the sulfur atom of CoA, could serve as whicha general is where acid the in acceptor catalysis. substrateKinetic analysis is thought with the to bind.physiological Structural substrate analysis (spermine) of BsPaiA showed showed that that the side chain of Y142, that interacts with the sulfur atom of CoA, could serve as a general acid in catalysis. Kinetic analysis with the physiological substrate (spermine) showed that the enzyme follows Int. J. Mol. Sci. 2016, 17, 1018 19 of 45

Michaelis–MentenInt. J. Mol. Sci. 2016, kinetics17, 1018 with an apparent Km of 76 µM, a Vmax of 480 nmol/min/mg enzyme19 of 44 and ´1 a kcat of 19.1 min [126]. theAnalysis enzyme of thefollows crystal Michaelis–Menten structures of SSAT kinetics homologs with an from apparent other sourcesKm of 76 including µM, a V human,max of 480 mouse, nmol/min/mg enzyme and a kcat of 19.1 min−1 [126]. Vibrio cholerae and Thermoplasma acidophilum, revealed different oligomeric forms [127–130]. SSATs from Analysis of the crystal structures of SSAT homologs from other sources including human, human (hSSAT) and mouse (Mus musculus) (MmSSAT) form a dimer in the crystal by interchanging the mouse, Vibrio cholerae and Thermoplasma acidophilum, revealed different oligomeric forms [127–130]. β7 strandsSSATs betweenfrom human the (hSSAT) two monomers. and mouse Interestingly, (Mus musculus the) SSAT(MmSSAT) homolog form froma dimerV. choleraein the crystal(SpeG) by exists as a dodecamerinterchanging (dimer the β of7 strands hexamers) between both the in solutiontwo monomers. and in theInterestingly, crystal [127 the]. However,SSAT homolog each from monomer of theV. hexamercholerae (SpeG) forms exists a dimer as a dodecamer with a neighboring (dimer of hexame monomerrs) both from in solution the second and in the hexamer. crystal [127]. The way the twoHowever, monomers each monomer associate of into the the hexamer dimer forms in SpeG a dimer is different with a toneighboring the SSAT homologsmonomer from from the human and mouse,second hexamer. but similar The toway many the two GNAT monomers enzymes associate from into other the families,dimer in SpeG including is different EfAAC(6 to the1 SSAT)-Ii [13 ,34] and StRimLhomologs [10 from]. The human SpeG and dodecamer mouse, but has similar six allosteric to many sites GNAT and enzymes six active from sites other [131 ].families, Binding of a polyamineincluding molecule EfAAC(6 (spermidine/spermine)′)-Ii [13,34] and StRimL [10]. to The the allostericSpeG dodecamer site of SpeGhas six induces allosteric structural sites and changessix active sites [131]. Binding of a polyamine molecule (spermidine/spermine) to the allosteric site of that result in binding of AcCoA and an additional polyamine molecule to the active site [131]. SpeG induces structural changes that result in binding of AcCoA and an additional polyamine The bell-shaped k pH profile of hSSAT is consistent with the involvement of both a catalytic molecule to the activecat site [131]. acid (with a pK value of 8.9) and a catalytic base (with pK of 7.3), whose ionization is crucial for The bell-shapeda kcat pH profile of hSSAT is consistent witha the involvement of both a catalytic catalysisacid [(with132]. a Detailed pKa value structural of 8.9) and analysis a catalytic of base the hSSAT/AcCoA(with pKa of 7.3), binarywhose ionization complex revealedis crucial for that the sulfurcatalysis atom of[132]. AcCoA Detailed is withinstructural a hydrogenanalysis of bondingthe hSSAT/AcCoA distance binary from complex the conserved revealed residue that the Y140, whichsulfur serves atom as of a AcCoA general is acidwithin in a catalysishydrogen [bonding132]. Substitution distance from of the Y140 conserved with phenylalanineresidue Y140, which lowered the enzyme’sserves as a activity general toacid less in thancatalysis 5% [132]. of the Substitution wild-type of and Y140 thus with confirmed phenylalanine its role lowered as a catalyticthe residueenzyme’s [133,134 activity]. Analysis to less of than hSSAT 5% inof complexthe wild-t withype theand bisubstratethus confirmedN1-spermine-AcCoA its role as a catalytic showed 1 that E92residue could [133,134]. serve Analysis as a remote of hSSA generalT in complex base to with perform the bisubstrate water-mediated N -spermine-AcCoA proton extraction showed from that E92 could serve as a remote general base to perform water-mediated proton extraction from spermine [132]. Substitution of E92 with glutamine in the mouse homolog MmSSAT confirmed spermine [132]. Substitution of E92 with glutamine in the mouse homolog MmSSAT confirmed the the essential role of E92 in the reaction [128]. An ordered sequential mechanism for acetyl transfer essential role of E92 in the reaction [128]. An ordered sequential mechanism for acetyl transfer (polyamine(polyamine binds binds first, first, followed followed by by AcCoA) AcCoA) was was observed observed in in rat rat liver liver SSAT SSAT [[128],128], whereaswhereas BsPaiA BsPaiA and hSSATand follow hSSAT a follow random-order a random-order ternary ternary complex complex mechanism mechanism [126 [126].].

Figure 13. Cartoon representation of the structure of Bacillus subtilis PaiA in complex with an oxidized Figure 13. Cartoon representation of the structure of Bacillus subtilis PaiA in complex with an oxidized CoA dimer (PDB ID: 1TIQ [126]). The conserved and non-conserved motifs of the GNAT domain are CoA dimer (PDB ID: 1TIQ [126]). The conserved and non-conserved motifs of the GNAT domain are colored as in Figure 3. colored as in Figure3. 2.12. C-Terminal Nε-Lysine Protein Acetyltransferase Family (EC 2.3.1.-) 2.12. C-Terminal Nε-Lysine Protein Acetyltransferase Family (EC 2.3.1.-) Nε-lysine acetylation of proteins occurs in all domains of life [135]. Lysine acetylation contributes toNε the-lysine control acetylation of gene expression of proteins in eukaryotes, occurs in all archaea domains and ofsome life bacteria. [135]. Lysine It plays acetylation an important contributes role to thein control bacterial of metabolism gene expression and other in eukaryotes,aspects of cell archaea physiology and [135,136]. some bacteria. It plays an important role ε in bacterialPA4794 metabolism (160 aa) and is otherC-terminal aspects N -lysine of cell physiologyprotein acetyltransferase [135,136]. from P. aeruginosa that catalyzes both N- and O-acetylation of proteins or small molecules, including chloramphenicol [137]. PA4794 (160 aa) is C-terminal Nε-lysine protein acetyltransferase from P. aeruginosa that catalyzes PA4794, the structure of which is shown in Figure 14A, is monomeric both in solution and in the bothcrystalN- and [137].O-acetylation It contains a of conserved proteins V-shaped or small cleft molecules, between includingthe β4 and chloramphenicolβ5 strands, a typical [ 137“P-loop”]. PA4794, the structureat the N-terminal of which end is shownof the α in3 helix Figure (R88-G89-L90-G91-V92-A93), 14A, is monomeric both inand solution a β-bulge and at inA77 the in crystal the β4 [137]. It containsstrand. aStructural conserved analysis V-shaped of the cleftPA4794/AcCoA between thebinaryβ4 complex and β5 revealed strands, that a typical a tyrosine “P-loop” residue at the N-terminal(Y128) is end positioned of the α near3 helix the (R88-G89-L90-G91-V92-A93),thioester sulfur atom of AcCoA, and can a β -bulgetherefore at serve A77 inas thea generalβ4 strand. Structural analysis of the PA4794/AcCoA binary complex revealed that a tyrosine residue (Y128) is positioned near the thioester sulfur atom of AcCoA, and can therefore serve as a general acid Int. J. Mol. Sci. 2016, 17, 1018 20 of 45 by donating a proton to the leaving thiolate anion of CoA. The thioester oxygen of AcCoA forms a hydrogenInt. J. Mol.bond Sci. 2016 with, 17, 1018 the backbone amide of M81 and thus contributes to the proper positioning20 of 44 and stabilization of AcCoA. Analysis of the ternary complex of PA4794 with CoA and acetylated acid by donating a proton to the leaving thiolate anion of CoA. The thioester oxygen of AcCoA forms peptide N-phenylacetyl-Gly-Lys revealed the role of the C-terminal free carboxyl group of the peptide a hydrogen bond with the backbone amide of M81 and thus contributes to the proper positioning in promoting the acetylation of lysine. There was no appropriately oriented conserved residue in and stabilization of AcCoA. Analysis of the ternary complex of PA4794 with CoA and acetylated the structurepeptide N that-phenylacetyl-Gly-Lys could be a candidate revealed for the a general role of base.the C-terminal However, free several carboxyl well-ordered group of the water moleculespeptide are in presentpromoting in thethe activeacetylation site thatof lysine. form There interactions was no withappropriately the main oriented chain peptide conserved groups of Y28residue andF118, in the andstructure theside that chaincould be of a N121, candidate and for that a general can mediate base. However, extraction several of a well-ordered proton from the primarywater amine molecules of an are acceptor present substrate. in the active The site Y128F that andform Y128A interactions variants with of the PA4794 main werechain catalyticallypeptide inactive,groups which of Y28 confirmed and F118, itsand essential the side chain role of in N121, the reaction. and that can Furthermore, mediate extraction substitution of a proton of N121 from with Ala resultedthe primary in a amine significant of an decreaseacceptor insubstrate. the enzyme The Y128F activity and [137 Y128A], confirming variants of its PA4794 role in were catalysis. In addition,catalytically structural inactive, analysis which confirmed of PA4794 its inessential complex role with in the antibiotics reaction. Furthermore, showed that substitution cephalosporins of N121 with Ala resulted in a significant decrease in the enzyme activity [137], confirming its role in occupy the substrate-binding site and thus act as competitive inhibitors [137]. catalysis. In addition, structural analysis of PA4794 in complex with antibiotics showed that Rv1347c (210 aa) is lysine Nε-acyltransferase of M. tuberculosis. It is essential for the mycobacterial cephalosporins occupy the substrate-binding site and thus act as competitive inhibitors [137]. survival withinRv1347c infected (210 aa) cells is lysine and N contributesε-acyltransferase to virulence of M. tuberculosis [138]. Rv1347c. It is essential is involved for the in mycobacterial the biosynthesis of thesurvival mycobactin within siderophoreinfected cells in andM. contributes tuberculosis to. Itvirulence catalyzes [138]. acetylation Rv1347c is of involved one or bothin the of the Nε-hydroxylysinebiosynthesis of armsthe mycobactin of mycobactin siderophore [138]. in The M. biosynthesistuberculosis. It ofcatalyzes siderophores acetylation was of also one linkedor both with pathogenesisof the Nε-hydroxylysine in other bacteria arms[ of139 mycobactin–142]. Structural [138]. The analysis biosynthesis revealed of siderophores that Rv1347c was also crystallizes linked as a monomerwith pathogenesis [138]. It hasin other a V-shaped bacteria [139–142]. AcCoA bindingStructural site analysis and anrevealed atypical that “P-loop”.Rv1347c crystallizes Rv1347c has no β-bulgeas a monomer in the [138].β4 strand. It has a V-shaped However, AcCoA it harbors binding a site catalytically and an atypical important “P-loop”. H130 Rv1347c residue has no at the correspondingβ-bulge in βthe-bulge β4 strand. position However, [143]. It it has harbors an N-terminal a catalytically extension important that servesH130 residue as a cap at on the top of corresponding β-bulge position [143]. It has an N-terminal extension that serves as a cap on top of the substrate binding cavity (Figure 14B). Another deviation from the conserved core GNAT fold the substrate binding cavity (Figure 14B). Another deviation from the conserved core GNAT fold in in Rv1347c is that the β7 strand is positioned between the β5 and β6 strands. A kinetic study with Rv1347c is that the β7 strand is positioned between the β5 and β6 strands. A kinetic study with a a bisubstratebisubstrate inhibitor inhibitor and and pHpH dependencedependence assays assays showed showed that that Rv1347c Rv1347c follows follows a random-order a random-order ternaryternary complex complex mechanism mechanism in in which which H130 H130 servesserves as a a general general base. base. D168 D168 was was shown shown to be to also be also importantimportant for catalysis, for catalysis, although although its its specific specific role role remainsremains to to be be established established [143]. [143 ].

ε FigureFigure 14. 14.Cartoon Cartoon representationrepresentation ofof the the structure structure of N of-lysineNε-lysine protein protein acetyltransferase. acetyltransferase. (A) (A) PseudomonasPseudomonas aeruginosaaeruginosa PA4794PA4794 in in complex complex with with AcCoA AcCoA (PDB (PDB ID: 3PGP ID: 3PGP [137]); [ 137and]); (B and) Rv1347c (B) Rv1347c of M. of tuberculosis (PDB IS: 1NYK3 [138]). The conserved and non-conserved motifs of the GNAT domains M. tuberculosis (PDB IS: 1NYK3 [138]). The conserved and non-conserved motifs of the GNAT domains are colored as in Figure 3. are colored as in Figure3.

Int. J. Mol. Sci. 2016, 17, 1018 21 of 45

2.13. Ribosomal Protein Nα-Acetyltransferase Family (EC 2.3.1.128) Int.Nα J.-acetylation Mol. Sci. 2016, 17 of, 1018 proteins is a common phenomenon in eukaryotes, where it plays an21 important of 44 α role in2.13. the Ribosomal control ofProtein proteins Nα-Acetyltransferase function and stabilityFamily (EC [144 2.3.1.128)]. In prokaryotes, where N -acetylation is less common, transferases RimI, RimJ, and RimL acetylate the α-amino group of N-terminal amino acids Nα-acetylation of proteins is a common phenomenon in eukaryotes, where it plays an important in ribosomal proteins S18, S5 and L12, respectively [145,146]. role in the control of proteins function and stability [144]. In prokaryotes, where Nα-acetylation is less S. typhimurium common,RimI (148 transferases aa) of RimI, RimJ, and(StRimI) RimL existsacetylate as athe monomer α-amino ingroup solution; of N-terminal the monomers amino acids assemble intoin a trimerribosomal in proteins the crystal, S18, S5 but and this L12, trimer respectively does [145,146]. not appear to be physiologically relevant [147]. StructuralRimI analysis (148 aa) revealed of S. typhimurium that strands (StRimI)β4 exists and βas5 aof monomer the core inβ solution;-sheetform the monomers a V-shaped assemble cleft, and the βinto7 strand a trimer is positionedin the crystal, between but this the trimerβ5 anddoes βnot6 strands appear to (Figure be physiologically 15A). StRimI relevant has a conserved[147]. “P-loop”Structural Q76-R77-R78-G79-L80-G81 analysis revealed that strands between β4 and the β5 βof4 the strand core β and-sheet the formα3 helixa V-shaped that interacts cleft, and withthe the pyrophosphateβ7 strand is positioned moiety of between AcCoA. the The β5 and carbonyl β6 strands moiety (Figure of 15A). AcCoA StRimI forms has a aconserved hydrogen “P-loop” bond with the main-chainQ76-R77-R78-G79-L80-G81 NH group of between I69 that the is thoughtβ4 strand to and polarize the α the3 helix acetyl that group interacts and, with in addition,the playpyrophosphate an important moiety role in of stabilizingAcCoA. The the carbonyl tetrahedral moiety reactionof AcCoA intermediate. forms a hydrogen Detailed bond with analysis the of main-chain NH group of I69 that is thought to polarize the acetyl group and, in addition, play an the structures of the StRimI/AcCoA and StRimI/bisubstrate analog complexes revealed that Y115 important role in stabilizing the tetrahedral reaction intermediate. Detailed analysis of the structures formsof athe hydrogen StRimI/AcCoA bond and with StRimI/bisubstrate the sulfur atom analog of AcCoA, complexes and revealed could that therefore Y115 forms serve a hydrogen as a general acidbond that donateswith the sulfur a proton atom toof AcCoA, the leaving and could group. therefore Furthermore, serve as a general the structures acid that suggesteddonates a proton that E103 can serveto the asleaving a general group. base Furthermore, that extracts the structures a proton su fromggested the that primary E103 can amine serve of as thea general acceptor base substratethat via aextracts well-ordered a proton waterfrom the molecule. primary amine Kinetic of the analysis acceptor showed substrate that via a StRimI well-ordered follows water adirect molecule. ordered nucleophilicKinetic analysis addition-elimination showed that StRimI mechanism follows wherea direct AcCoA ordered binds nucleophilic first, followed addition-elimination by the acceptor substratemechanism [147]. where AcCoA binds first, followed by the acceptor substrate [147].

FigureFigure 15. 15.Cartoon Cartoon representation representation of of the the structures structures of ribosomal of ribosomal protein protein Nα- acetyltransferases.Nα- acetyltransferases. (A). (A). SalmonellaSalmonella typhimurium typhimurium RimIRimI in complex in complex with witha bisubstrate a bisubstrate inhibitor, inhibitor, C-term-Arg-Arg-Phe-Tyr-Arg- C-term-Arg-Arg-Phe-Tyr- Ala-N-α-AcCoA (PDB ID: 2CNM [147]); and (B) RimL from S. typhimurium in complex with CoA Arg-Ala-N-α-AcCoA (PDB ID: 2CNM [147]); and (B) RimL from S. typhimurium in complex with CoA (PDB ID: 1S7N [108]). The conserved and non-conserved motifs of the GNAT domains are colored as (PDB ID: 1S7N [108]). The conserved and non-conserved motifs of the GNAT domains are colored as in Figure 3. in Figure3.

Int. J. Mol. Sci. 2016, 17, 1018 22 of 45

RimL (179 aa) of S. typhimurium (StRimL) is responsible for converting the prokaryotic ribosomal protein L12 into its acetylated form (L7) by transferring an acetyl group from AcCoA onto the N-terminal amino group of L12 [108]. StRimL also possesses the MccE activity [148]. It is dimeric both Int. J. Mol. Sci. 2016, 17, 1018 22 of 44 in solution and in the crystal. Structural analysis revealed that the β4 and β5 strands of StRimL are splayed apartRimL (179 to create aa) of aS. V-shapedtyphimurium groove (StRimL) for is AcCoA responsible binding for converting (Figure the15B). prokaryotic The “P-loop” ribosomal between the βprotein4 strand L12 and into the itsα acetylated3 helix interacts form (L7) with by thetransferring diphosphate an acetyl group group of CoA. from The AcCoA StRimL onto structurethe containsN-terminal no β-bulge amino in group the active of L12 site [108]. [108 StRimL]. The alsoβ7 strandpossesses is placedthe MccE between activity the[148].β5 It and is dimericβ6 strands, both and the βin-strands solution at and the in dimer the crystal. interface Structural are arranged analysis revealed to form athat continuous the β4 andβ β-sheet.5 strands The of dimerStRimL interfaceare containssplayed the proteinapart to substratecreate a V-shaped binding groove site. Kinetic for AcCoA analysis binding showed (Figure that 15B). StRimL The “P-loop” follows abetween direct acetyl transferthe β mechanism4 strand and [ 108the ].α3 The helix carbonyl interacts oxygen with the of diphosphate AcCoA is hydrogen-bondedgroup of CoA. The StRimL with the structure main-chain contains no β-bulge in the active site [108]. The β7 strand is placed between the β5 and β6 strands, amide of Y98 that could serve for the proper positioning of the acetyl group and for the polarization of and the β-strands at the dimer interface are arranged to form a continuous β-sheet. The dimer the carbonyl carbon, and thereby stabilization of the tetrahedral reaction intermediate. S141 is within interface contains the protein substrate binding site. Kinetic analysis showed that StRimL follows a hydrogen-bondingdirect acetyl transfer distance mechanism from the [108]. sulfur The atomcarbonyl of boundoxygen CoA,of AcCoA and is can hydrogen-bonded therefore serve with as a the general acidmain-chain in catalysis. amide E160 of is Y98 appropriately that could serve positioned for the pr tooper act positioning as a general of the base acetyl to extractgroup and a proton for the from the αpolarization-amino group of the of carbonyl L12 as carbon, the first and reaction thereby stepstabilization [108]. Theof the structures tetrahedral of reaction RimL intermediate. and its homolog fromS141B. subtilis is within, YadF, hydrogen-bonding are very similar distance [149]. However,from the sulfur in contrast atom of to bound the dimeric CoA, and RimL, can YadFtherefore exists as a mixtureserve as of a dimers general and acid hexamers in catalysis. in E160 solution is appropriately and crystallizes positioned as a to trimer act as ofa general dimers. base to extract a proton from the α-amino group of L12 as the first reaction step [108]. The structures of RimL and 2.14.its Succinyltransferase homolog from B. subtilis Family, YadF, (EC 2.8.3.-) are very similar [149]. However, in contrast to the dimeric RimL, YadF exists as a mixture of dimers and hexamers in solution and crystallizes as a trimer of dimers.2.14.Rv0802c of M.Succinyltransferase tuberculosis (MtRv0802c, Family (EC 218 2.8.3.-) aa) is a putative succinyltransferase that is thought to utilize succinylRv0802c CoA of M. (SucCoA), tuberculosis rather (MtRv0802c, than AcCoA, 218 aa) is as a anputative acyl donor succinyltransferase [4]. Structural that and is thought biophysical analysisto utilize revealed succinyl that CoA MtRv0802c (SucCoA), existsrather asthan a tetramerAcCoA, as (dimer an acyl donor of dimers) [4]. Structural both in and solution biophysical and in the crystal,analysis as is therevealed case forthat yeast MtRv0802c Hpa2 [exists4,76]. as Each a tetram monomerer (dimer of MtRv0802cof dimers) both accommodates in solution and one in molecule the of SucCoA.crystal, as It hasis the a case “P-loop” for yeast between Hpa2 [4,76]. the β Ea4 strandch monomer and the of MtRv0802cα3 helix that accommodates forms hydrogen one molecule bonds with the pyrophosphateof SucCoA. It has moiety a “P-loop” of SucCoA. between Thethe β SucCoA4 strand and binds the in α3 the helix V-shaped that forms cleft hydrogen between bonds the β with4 and β5 strandsthe (Figurepyrophosphate 16). MtRv0802c moiety of SucCoA. has a C-terminal The SucC extensionoA binds in (two the V-shapedα-helices cleft and between a β-strand), the β4 not and found β5 strands (Figure 16). MtRv0802c has a C-terminal extension (two α-helices and a β-strand), not in other GNAT proteins. However, the function of this extension is not yet known. Detailed analysis found in other GNAT proteins. However, the function of this extension is not yet known. Detailed showed that the carbonyl oxygen of the succinyl group forms a hydrogen bond with the main-chain analysis showed that the carbonyl oxygen of the succinyl group forms a hydrogen bond with the β amidemain-chain of S111 onamide the of4 S111 strand. on the The β4 hydrogen strand. The bond hydrogen between bond the between carbonyl the groupcarbonyl of group SucCoA of and the main-chainSucCoA and amidethe main-chain of a residue amide onof a theresidueβ4 strandon the β is4 strand a conserved is a conserved feature feature found found in otherin other GNAT proteinsGNAT [10 proteins]. [10].

FigureFigure 16. 16.Cartoon Cartoon representation representation ofof thethe structurestructure of of putative putative M.M. tuberculosis tuberculosis succinyltransferasesuccinyltransferase (Rv0802c) in complex with succinyl CoA (PDB ID: 2VZZ [4]). The conserved and non-conserved (Rv0802c) in complex with succinyl CoA (PDB ID: 2VZZ [4]). The conserved and non-conserved motifs motifs of the GNAT domain are colored as in Figure 3. of the GNAT domain are colored as in Figure3. 2.15. FemABX Aminoacyl Transferases Family (FemABX, EC 2.3.2.-) 2.15. FemABX Aminoacyl Transferases Family (FemABX, EC 2.3.2.-) Members of the FemABX (factors essential for methicillin resistance) family are involved in the synthesisMembers of of peptide the FemABX bridges that (factors crosslink essential peptides for in methicillin peptidoglycan resistance) in some familybacteria. are They involved transfer in the synthesisan aminoacyl of peptide group bridges from aminoacyl-tRNA that crosslink peptides to the ε-amino in peptidoglycan group of Lys or in meso some-diaminopimelic bacteria. They acid transfer

Int. J. Mol. Sci. 2016, 17, 1018 23 of 45 an aminoacyl group from aminoacyl-tRNA to the ε-amino group of Lys or meso-diaminopimelic acid at position 3 of the peptidoglycan precursor, which can be either UDP-MurNAc-pentapeptide, or lipid II [150–152]. They play an important role in maintaining the peptidoglycan structure and thus contribute both to the bacterial viability and methicillin resistance [153,154]. FemX adds the first residue, FemA catalyzes the addition of the second and third residue (typically glycine) to the growing pentaglycine interpeptide, while FemB incorporates the fourth and fifth glycine [151]. Insertional inactivation of the femAB operon resulted in altered glycine composition in the bacterial cell wall [155]. The femAB knock-out strain showed absolute sensitivity to methicillin and, in addition, became hypersensitive to other types of antimicrobial agents [156]. Structural analysis of FemA of Staphylococcus aureus (SaFemA, EC 2.3.2.17) revealed that SaFemA is monomeric in the crystal and is composed of two GNAT domains (domain 1: residues 1–144 and domain 2: residues 145–395). Domain 2 contains a long coiled subdomain consisting of a pair of antiparallel α-helices (residues 246–307) inserted between the β3 and β4 strands (Figure 17A) [150]. The presence of this helical “arm” is a unique structural feature of SaFemA. Comparisons with bacterial seryl-tRNA synthetase suggested that the helical arm forms the glycyl-tRNA substrate binding site [150,157]. The two GNAT domains together form an L-shaped channel that traverses SaFemA and is thought to accommodate a binding site for a peptidoglycan precursor. This is an interesting example of an enzyme that utilizes the GNAT fold to catalyze a reaction that does not involve AcCoA. FemX from Weissella viridescens (WvFemX, EC 2.3.2.10) catalyzes the transfer of L-Ala to the side-chain amino group of Lys present at the third position of the W. viridescens peptidoglycan precursor UDP-MurNAc-pentapeptide, using Ala-tRNAAla as a donor substrate. Structural analysis of WvFemX in the free form and in complex with the acceptor substrate (UDP-MurNAc-pentapeptide) or reaction product (UDP-MurNAc-pentapeptide-Ala) revealed that WvFemX forms a dimer in the crystal [158]. The WvFemX monomer harbors two GNAT domains: an N-terminal (residues 1–145 and 317–335) and a C-terminal (residues 146–316) one. The overall topology of WvFemX is similar to that of SaFemA, except for the helical arm that is absent in WvFemX (Figure 17B). The N-terminal GNAT domain of WvFemX interacts with UDP-MurNAc-pentapeptide, while the C-terminal GNAT domain interacts with the donor Ala-tRNA. Site-directed mutagenesis confirmed the important roles of K36, R211 and Y215 for the substrate binding [159]. The orientation of the aromatic ring and the hydroxyl group of Y254 (β10 strand) suggested its involvement in the tRNA binding [152]. K305 and F304 were shown to be essential for the catalytic activity [158]. K305 could contribute to the stabilization of the negative charge that develops on the carbonyl oxygen of L-Ala after the nucleophilic attack of the ester bond of Ala-tRNAAla by the amine group of UM5P [152]. Interestingly, in contrast to the GNAT acetyltransferases, FemX aminoacyl transferase follows a novel substrate-aided transfer mechanism rather than using a general catalytic base or acid [158]. Leucyl/phenylalanyl-tRNA protein transferase of E. coli (EcLFT, EC 2.3.2.6) shows significant topological and structural similarities to FemABX enzymes, although it acts on a different substrate. It catalyzes the transfer of leucine or phenylalanine (and, to a lesser extent, methionine or tryptophan) to the N-terminal Arg or Lys residue of proteins, using Leu-tRNALeu or Phe-tRNAPhe as the second substrate [152,160,161]. It plays an important role in protein degradation [161]. The structural analysis of EcLFT in complex with an aminoacyl-tRNA analog puromycin showed that the EcLFT monomer has a C-terminal GNAT domain comprising residues 63 to 232 [161] (Figure 17C) that has a similar topology to the C-terminal GNAT domain of WvFemX and SaFemA. The N-terminal domain of EcLFT has a different, non-GNAT, fold. The 6-N,N-dimethyladenine moiety mimicking the 31-terminal adenosine of aminoacyl-tRNAs is stabilized by a π–π stacking interaction with W49 from the N-terminal domain, which suggests that the N-terminal domain interacts with the acceptor substrate. The p-methoxybenzyl moiety of puromycin, mimicking the amino-acid moiety of Leu-tRNALeu or Phe-tRNAPhe, is harbored in a highly hydrophobic pocket formed by residues M144, F153, L170, F173 and I185. Mutations of the residues that form stabilizing interactions with puromycin significantly decreased the activity of Int. J. Mol. Sci. 2016, 17, 1018 24 of 45

EcLFT, confirming their important role in the recognition of aminoacyl-tRNAs. Analysis of the model of the EcLFTInt. J. Mol. complex Sci. 2016, 17 with, 1018 tRNA and a substrate protein suggested that the side chains24 of of E15644 and Q188 serve to anchor the positively charged side chain of the N-terminal Arg or Lys of the acceptor the N-terminal Arg or Lys of the acceptor protein close to the aminoacyl bond of the aminoacyl-tRNA proteinto close promote to the the aminoacyl formation of bond the peptide of the aminoacyl-tRNAbond. These residues to are promote therefore the thought formation to define of the thepeptide bond. Theseacceptor residues substrate are spec thereforeificity of EcLFT thought [161]. to define the acceptor substrate specificity of EcLFT [161].

Figure 17.FigureCartoon 17. Cartoon representation representation of of the the structuresstructures of of aminoacyl aminoacyl transferas transferaseses from fromthe FemABX the FemABX family.family. (A) Staphylococcus (A) Staphylococcus aureus aureusFemA FemA apoenzyme apoenzyme (PDB (PDB ID: ID: 1LRZ 1LRZ [150]); [150 (B]);) Weissella (B) Weissella viridescens viridescens FemX in complex with UDP-MurNAc-pentapeptide (UM5P) substrate (PDB ID: 1P4N [152]); and (C) FemX in complex with UDP-MurNAc-pentapeptide (UM5P) substrate (PDB ID: 1P4N [152]); and E. coli leucyl/phenylalanyl-tRNA protein transferase (EcLFT) in complex with puromycin (PDB ID: (C) E. coli2DPTleucyl/phenylalanyl-tRNA [161]). The conserved and proteinnon-conserved transferase motifs (EcLFT) of the GNAT in complex domains with are puromycincolored as in (PDB ID: 2DPT [161Figure]). The 3. conserved and non-conserved motifs of the GNAT domains are colored as in Figure3.

2.16. Protein2.16. Protein N-Myristoyltransferase N-Myristoyltransferase Family Family (NMT, (NMT, ECEC 2.3.1.97) N-myristoyltransferase (NMT) facilitates the transfer of myristate (a 14-carbon saturated fatty N-myristoyltransferase (NMT) facilitates the transfer of myristate (a 14-carbon saturated acid) from myristoyl CoA (Myr-CoA) to the N-terminal glycine residue of various fungal, eukaryotic, fatty acid)protozoan from and myristoyl viral protei CoAns [162–167]. (Myr-CoA) Myristoyla to thetion N-terminal of proteins glycinecan promote residue reversible of various protein– fungal, eukaryotic,protein protozoan interactions, and interactions viral proteins between [162 proteins–167]. Myristoylationand the cellular membra of proteinsne, or can enhance promote protein reversible protein–proteinstability [165]. interactions, However, this interactions modification, between or its abnormal proteins levels, and was the cellularalso linked membrane, to many human or enhance proteindiseases stability including [165]. However,cancer, genetic this disorders modification, and viral or infection its abnormal [165]. levels, was also linked to many NMT (451 aa) from Candida albicans (CaNMT) is a monomeric enzyme that, like FemABX human diseases including cancer, genetic disorders and viral infection [165]. aminoacyl transferases, harbors two GNAT domains. It has an internal two-fold symmetry, likely as NMTa result (451 of aa)gene from duplication,Candida although albicans the (CaNMT)two domains is show a monomeric very limited enzymesequence identity that, like [168]. FemABX aminoacylStructural transferases, analysis harborsof its homolog two GNAT from S. domains. cerevisiae It(ScNMT) has an in internal complex two-fold with Myr-CoA symmetry, and a likely as a resultnon-peptide of gene duplication, inhibitor [3,169] although suggested the that two its domains N-terminal show region very (residues limited 4–30), sequence colored cyan identity in [168]. StructuralFigure analysis 18, plays of an itsimportant homolog role in from the recognitionS. cerevisiae of both(ScNMT) Myr-CoA inand complex the peptide with substrate. Myr-CoA The and a non-peptideC-terminal inhibitor GNAT [domain3,169] suggestedaccommodates that the its N-terminalpeptide substrate region binding (residues site, occupied 4–30), colored by the cyan in inhibitor molecule in the ternary complex, whilst the N-terminal GNAT domain accommodates Figure 18, plays an important role in the recognition of both Myr-CoA and the peptide substrate. The Myr-CoA [169]. Comparison of the structures of the NMT homologs from yeast, human, fungi and C-terminalparasites GNAT (Table domain 1) revealed accommodates that while the the Myr-Co peptideA binding substrate site bindingis conserved, site, the occupied peptide substrate by the inhibitor moleculebinding in the site ternary differs complex, significantly whilst among the NMTs N-terminal from different GNAT species, domain which accommodates reflects their Myr-CoAdifferent [169]. Comparisonpeptide of substrate the structures specificities of the [170]. NMT In homologscommon with from GNAT yeast, acetyltransferases, human, fungi andNMT parasites follows an (Table 1) revealedordered that while Bi-Bi thecatalytic Myr-CoA reaction binding mechanism site is[163] conserved,. The backbone the peptide amides substrateof both F170 binding and L171 site differs significantly among NMTs from different species, which reflects their different peptide substrate specificities [170]. In common with GNAT acetyltransferases, NMT follows an ordered Bi-Bi catalytic reaction mechanism [163]. The backbone amides of both F170 and L171 generate the oxyanion hole Int. J. Mol. Sci. 2016, 17, 1018 25 of 45

Int. J. Mol. Sci. 2016, 17, 1018 25 of 44 that polarizes the thioester carbonyl moiety of Myr-CoA and contributes to the stabilization of the tetrahedralgenerate intermediate the oxyanion hole generated that polarizes during the the thioester transfer carbonyl reaction moiety [3]. of The Myr-CoA carboxylate and contributes moiety of the C-terminalto the stabilization residue L455 of the would tetrahedral be within intermediate hydrogen generated bonding during distance the transfer from thereaction amino [3]. groupThe of the N-terminalcarboxylate moiety glycine of of the the C-terminal peptide residue substrate, L455 indicating would be within that L455hydrogen could bonding serve distance as a general from base the amino group of the N-terminal glycine of the peptide substrate, indicating that L455 could serve deprotonating the substrate in the first reaction step. Mutagenesis studies confirmed the essential as a general base deprotonating the substrate in the first reaction step. Mutagenesis studies confirmed role ofthe L455 essential in catalysis role of L455 [171 in]. catalysis Within [171]. the GNAT Within superfamily,the GNAT superfamily, the involvement the involvement of the C-terminalof the carboxylateC-terminal group carboxylate in catalysis group is in unique catalysis to is NMT unique [172 to]. NMT Structures [172]. Structures of CaNMT of CaNMT homologs homologs from other sources,from including other sources,Plasmodium including[173 Plasmodium–177], Leishmania [173–177],[178 Leishmania–181], Treponema [178–181],[182 Treponema], fungus [182], [183] and humanfungus [184 [183]] have and also human been [184] reported have al inso literature. been reported in literature.

FigureFigure 18. 18.Cartoon Cartoonrepresentation representation of of the the structure structure of yeast ofyeast N-myristoyltransferaseN-myristoyltransferase in complex in with complex withmyristoyl myristoyl CoA CoA and and the non-peptidenon-peptide inhibitor ( ZZ)-3-benzyl-5-(2-hydroxy-3-nitrobenzylidene)-2-)-3-benzyl-5-(2-hydroxy-3-nitrobenzylidene)-2- thioxothiazolidin-4-one (PDB ID: 2P6F [169]). The conserved and non-conserved motifs of the GNAT thioxothiazolidin-4-one (PDB ID: 2P6F [169]). The conserved and non-conserved motifs of the GNAT domain, cofactor and inhibitor are colored as in Figure 3. domain, cofactor and inhibitor are colored as in Figure3. 2.17. Mycothiol Synthase Family (MshD, EC 2.3.1.189) 2.17. Mycothiol Synthase Family (MshD, EC 2.3.1.189) Mycothiol synthase (MshD,MW 33.6 kDa), encoded by the Rv0819 gene of M. tuberculosis, Mycothiolcatalyzes the synthase final step (MshD, in the MbiosynthesisW 33.6 kDa), of low encoded molecular by theweightRv0819 organosulfurgene of M.compound tuberculosis , catalyzesmycothiol the (MSH) final stepfrom 1- inL-myo-inositol-1-phosphate the biosynthesis of low [185]. molecular It acetylates weight the cysteinyl organosulfur amino group compound of mycothiolL-cysteine-1- (MSH)D-myo-inosityl-2-amido-2-deoxy- from 1-L-myo-inositol-1-phosphateα-D-glucopyranoside [185]. It acetylates (Cys-Gln-Ins) the cysteinyl using aminoAcCoA group as of an acetyl donor to generate MSH (AcCys-Gln-Ins). MSH is the major thiol compound in Mycobacteria L-cysteine-1-D-myo-inosityl-2-amido-2-deoxy-α-D-glucopyranoside (Cys-Gln-Ins) using AcCoA as and in most actinomycetes. It plays a significant role in neutralization of electrophiles, oxidative an acetyl donor to generate MSH (AcCys-Gln-Ins). MSH is the major thiol compound in Mycobacteria stress, antibiotic resistance and oxidation of formaldehyde in M. tuberculosis [185]. and in mostMshD actinomycetes. exists as a monomer It plays both a significant in solution role and in in neutralization the crystal [186,187]. of electrophiles, Structural oxidativeanalysis of stress, antibioticMshD resistance in complex and with oxidation AcCoA revealed of formaldehyde that this enzyme in M. tuberculosis harbors two[185 GNAT]. domains; residues MshD1–140 form exists the as aN-terminal monomer GNAT both indomain solution while and residues in the crystal 151–315 [186 form,187 ].the Structural C-terminal analysis one of MshD(Figure in complex 19). The withpyrophosphate AcCoArevealed binding “P-loop” that this an enzymed the V-shaped harbors AcCoA two binding GNAT cleft domains; are present residues 1–140in form both thedomains. N-terminal However, GNAT the structure domain suggested while residues that only 151–315 the C-terminal form the domain C-terminal is active, one because (Figure 19). The pyrophosphatein the N-terminal GNAT binding domain “P-loop” the acetyl and group the V-shaped of AcCoA AcCoAis positioned binding out of cleftreach are for the present acceptor in both domains.substrate. However, Analysis the of structurethe ternary suggested complex thatof MshD only thewith C-terminal CoA and domainthe natural is active, substrate because in thedesacetylmycothiol N-terminal GNAT (DAM) domain showed the that acetyl only one group DAM of molecule AcCoA binds is positioned in the central out cavity of reachbetween for the the two GNAT domains [187]. This indicates that the non-catalytic N-terminal domain provides acceptor substrate. Analysis of the ternary complex of MshD with CoA and the natural substrate residues that form part of the substrate-binding site, and is therefore important for function. Kinetic desacetylmycothiol (DAM) showed that only one DAM molecule binds in the central cavity between analysis of MshD showed a bell-shaped pH dependence of Vmax and V/KDAM, consistent with the the twoinvolvement GNAT domainsof a catalytic [187 base]. Thiswith p indicatesKa of 6.6–7.0 that and the a catalytic non-catalytic acid with N-terminal pK of 8.7–9.2 domain in the acetyl provides residuestransfer that reaction. form part Theof initial the velocity substrate-binding pattern of MshD site, confirmed and is therefore the direct important transfer of for thefunction. acetyl group Kinetic analysisfrom of AcCoA MshD to showed DAM via a bell-shapedformation of pHa tetrahedra dependencel-intermediate. of Vmax andThe Vstructure/KDAM ,showed consistent that withthe the involvementprimary amine of a catalytic moiety baseof DAM with is p positionedKa of 6.6–7.0 in an and orientation a catalytic that acid favors with the pK directof 8.7–9.2 nucleophilic in the acetyl transferattack reaction. on the Theacetyl initial group velocity of AcCoA pattern bound of MshDto the catalytically confirmed theactive direct C-terminal transfer GNAT of the domain. acetyl group fromStructural AcCoA toanalysis DAM revealed via formation that E234 of aat tetrahedral-intermediate. the C-terminal domain could The accept structure a proton showed from the that the primary amine of DAM via a well-ordered water molecule, and thus could serve as a general base. primary amine moiety of DAM is positioned in an orientation that favors the direct nucleophilic attack on the acetyl group of AcCoA bound to the catalytically active C-terminal GNAT domain. Structural analysis revealed that E234 at the C-terminal domain could accept a proton from the primary amine of Int. J. Mol. Sci. 2016, 17, 1018 26 of 45

DAMInt. via J. Mol. a well-ordered Sci. 2016, 17, 1018 water molecule, and thus could serve as a general base. The backbone26 of 44 amide nitrogen of L238 forms a hydrogen bond with the carbonyl oxygen to facilitate proper positioning of TheInt. J. backboneMol. Sci. 2016 amide, 17, 1018 nitrogen of L238 forms a hydrogen bond with the carbonyl oxygen to facilitate26 of 44 the acetyl group of AcCoA and could play an important role in stabilizing the tetrahedral reaction proper positioning of the acetyl group of AcCoA and could play an important role in stabilizing the intermediate.tetrahedralThe backbone The reaction amide hydroxyl intermediate. nitrogen group of ofL238 The Y294 formshydroxyl is positioneda hydrog groupen of nearbond Y294 towith is thepositioned the sulfur carbonyl atomnear oxygen to of the CoA sulfur to (~3.6facilitate atom Å) and couldofproper act CoA as positioning(~3.6 a general Å) and acid of could the to acetyl donateact as groupa general a proton of AcCoA acid to to the andonate leavingd could a proton play thiolate an to importantthe ion. leaving role thiolate in stabilizing ion. the tetrahedral reaction intermediate. The hydroxyl group of Y294 is positioned near to the sulfur atom of CoA (~3.6 Å) and could act as a general acid to donate a proton to the leaving thiolate ion.

FigureFigure 19. 19.Cartoon Cartoon representation representation ofof thethe structure of of M.M. tuberculosis tuberculosis MshDMshD in complex in complex with with desacetylmycothiol, AcCoA and CoA (PDB ID: 2C27 [187]). The conserved and non-conserved motifs desacetylmycothiol, AcCoA and CoA (PDB ID: 2C27 [187]). The conserved and non-conserved motifs ofFigure the GNAT 19. Cartoon domain, representation cofactor and subs of tratethe arestructure colored of as M.in Figuretuberculosis 3. MshD in complex with of the GNAT domain, cofactor and substrate are colored as in Figure3. desacetylmycothiol, AcCoA and CoA (PDB ID: 2C27 [187]). The conserved and non-conserved motifs 3. Oligomerizationof the GNAT domain, of GNAT cofactor Superfamily and substrate Enzymes are colored as in Figure 3. 3. Oligomerization of GNAT Superfamily Enzymes Previous studies on GNAT superfamily members suggested that many of them are oligomeric, 3. Oligomerization of GNAT Superfamily Enzymes Previousand that oligomerization studies on GNAT is important superfamily for their members function. suggested Most of the that GNAT many transferases of them are dimeric oligomeric, and that(Figure oligomerizationPrevious 20), althoughstudies on is examples importantGNAT superfamily of for monomeric their members function. (Figure suggested Most 20A), of that thetrimeric, many GNAT oftetrameric, transferasesthem are oligomeric,hexameric are dimeric (Figure(Figureand 20 that), although 20F) oligomerization and examplesdodecameric is important of monomeric enzymes for theirhave (Figure function.also 20beenA), Most trimeric,discussed of the tetrameric,GNATin this transferasesreview. hexameric Four are different dimeric (Figure 20F) (Figure 20), although examples of monomeric (Figure 20A), trimeric, tetrameric, hexameric and dodecamericdimerization modes enzymes have have been also observed. been discussed One of the in most this frequently review. Four occurring different arrangements dimerization of the modes β(Figure-strands 20F) at the and dimer dodecameric interface isenzymes where the have C-terminal also been β-strands discussed from in each this mo review.nomer Fourcome differenttogether have been observed. One of the most frequently occurring arrangements of the β-strands at the dimer todimerization form a continuous modes have β-sheet been (Figures observed. 20B One and of 21). th eThis most mode frequently of dimerization occurring wasarrangements observed inof the interface is where the C-terminal β-strands from each monomer come together to form a continuous crustalβ-strands structures at the dimer of interface StRimL, is whereEfAAC(6 the C-terminal)-Ii, HpPseH, β-strands MtRv0802c, from each mohSSATnomer and come EcWecDtogether β-sheet[10,107,108,113,126].to form (Figures a continuous 20B and In β 21the-sheet). second This (Figures modecommon 20B of and type dimerization 21). of dimersThis mode in was the of observedGNAT dimerization superfamily, in the was crustal observed the monomers structures in the of StRimL,exchangecrustal EfAAC(6)-Ii, structures their C-terminal HpPseH, of StRimL, β-strands MtRv0802c, EfAAC(6 so that hSSAT )-Ii,they HpPseH,are and inserted EcWecD MtRv0802c, in [the10 ,107core ,108 βhSSAT-sheet,113 , 126ofand the]. In oppositeEcWecD the second commonmonomer[10,107,108,113,126]. type of(Figures dimers 20C inIn the and second GNAT21). This common superfamily, type of type dimerization of the dimers monomers inwas the found GNAT exchange in su ScHpa2,perfamily, their SeAAC(6 C-terminal the monomers′)-Iyβ and-strands so thatScGNA1exchange they are [22,76,99].their inserted C-terminal The in thethird β-strands core typeβ -sheetsoof thatdimerizati of they the areon opposite insertedis exemplified monomer in the coreby (Figuresthe β-sheet crystal of20 Cthestructure and opposite 21 ).of This typeSmAAC(3)-Ia ofmonomer dimerization (Figures [5]. The was 20C two foundand SmAAC(3)-Ia 21). in This ScHpa2, type monomers of SeAAC(6 dimerization associate1)-Iy was andin such found ScGNA1 a way in ScHpa2, that [22 the,76 SeAAC(612,99 β].-strands The′)-Iy third form and type of dimerizationaScGNA1 β-barrel [22,76,99].at isthe exemplified dimer The interface third by (Figuretype the crystalof 20D). dimerizati structureThe fourthon is oftype exemplified SmAAC(3)-Ia of a dimer byis represented [the5]. Thecrystal two bystructure the SmAAC(3)-Ia crystal of SmAAC(3)-Ia [5]. The two SmAAC(3)-Ia monomers associate in such a way that the 12 β-strands form monomersstructure associate of hPCAF in where such the a way dimer that is stabilized the 12 βvia-strands formation form of a afour-helicalβ-barrel atbundle the dimerat the dimer interface interfacea β-barrel (Figure at the dimer20E). interface (Figure 20D). The fourth type of a dimer is represented by the crystal (Figurestructure 20D). Theof hPCAF fourth where type the of adimer dimer is isstabilized represented via formation by the crystalof a four-helical structure bundle of hPCAF at the wheredimer the dimerinterface is stabilized (Figure via 20E). formation of a four-helical bundle at the dimer interface (Figure 20E).

Figure 20. Cont.

Figure 20. Cont. Figure 20. Cont.

Int. J. Mol. Sci. 20162016,, 17,, 1018 1018 27 of 4544 Int. J. Mol. Sci. 2016, 17, 1018 27 of 44

Figure 20. Representative oligomeric structures observed in the GNAT superfamily. (A) Monomeric Figure 20. Representative oligomeric structures observed in the GNAT superfamily. (A)) MonomericMonomeric ScMpr1; (B) dimeric StRimL where the β-strands from the two monomers at the dimer interface are ScMpr1; (B) dimeric StRimL where thethe β-strands from the two monomers at the dimerdimer interfaceinterface areare arranged to form a continuous β-sheet; (C) dimeric interface where the C-terminal β-strands are arranged to form aa continuouscontinuous ββ-sheet;-sheet; (C) dimericdimeric interfaceinterface wherewhere thethe C-terminalC-terminal ββ-strands are interchanged between the monomers (ScGNA1); (D) a 12-strand β-barrel at the dimer interface of interchanged between the monomers (ScGNA1); (D) a 12-strand β-barrel at the dimer interface of interchangedSmAAC(3)-Ia; between (E) dimer the interface monomers involving (ScGNA1); α-helices (D from) a 12-strand both monomers-barrel (hPCAF); at the and dimer (F) one-half interface of SmAAC(3)-Ia; (E) dimer interface involving α-helices from both monomers (hPCAF); and (F) one-half SmAAC(3)-Ia;of the MtEis (E )hexamer dimer interface (two three-fold involving symmetricalα-helices trimers from both shown monomers in this Figure (hPCAF); assemble and into (F) one-halfan of the asymmetric MtEis MtEis hexamer hexamer “sandwich”). (two (two Thethree-fold three-fold conserved symmetrical symmetrical and non-co nservedtrimers trimers motifsshown shown of in the inthis GNAT this Figure Figuredomain, assemble assemble cofactors into into an anasymmetric asymmetricand substrates “sandwich”). “sandwich”). are colored The Theas conserved in conserved Figure 3. and and non-co non-conservednserved motifs motifs of of the the GNAT GNAT domain, domain, cofactors and substrates are coloredcolored asas inin FigureFigure3 3..

Figure 21. Schematic figure illustrating the topology of the β-strands found in the two most common types of dimer interface in GNAT proteins. Strands from one monomer are colored white, and those from the second monomer are colored gray. Figure 21. Schematic figurefigure illustrating the topology of the β-strands found in the two most common types4. General of dimer Catalytic interface Mechanism in GNAT of proteins.proteins. GNAT StrandsStrandSuperfamilys from oneMembers monomer are coloredcolored white,white, andand thosethose from thethe secondsecond monomermonomer areare coloredcolored gray.gray. Cumulatively, structural and kinetic studies on the GNAT enzymes and their complexes with substrates or inhibitors demonstrated that the members of this superfamily follow a common 4. General Catalytic Mechanism of GNAT Superfamily Members 4. Generalcatalytic Catalytic mechanism Mechanism that involves of a GNAT direct transfer Superfamily of an acyl Members group from a donor substrate (usually Cumulatively,AcCoA) on the amino structural group and of kinetican acceptor studies substr onate. the With GNAT the enzymesexceptionenzymes ofand Mpr1 theirtheir that complexescomplexes catalyzes with acetylation of secondary amines, all GNAT enzymes characterized to date act on primary amino substrates oror inhibitors inhibitors demonstrated demonstrated that that the membersthe members of this of superfamily this superfamily follow afollow common a common catalytic groups. Generally, the amino group of the acceptor substrate needs to be deprotonated to perform a mechanismcatalytic mechanism that involves that ainvolves direct transfer a direct of transfer an acyl groupof an acyl from group a donor from substrate a donor (usually substrate AcCoA) (usually on direct nucleophilic attack on the carbonyl carbon of the enzyme-bound AcCoA. Most GNAT enzymes AcCoA) on the amino group of an acceptor substrate. With the exception of Mpr1 that catalyzes the aminohave an group amino of acid an (usually acceptor Glu, substrate. Asp or Ser) With near the their exception active site of that Mpr1 serves that as catalyzes a general acetylationbase that of secondaryacetylationextracts amines, ofa protonsecondary all from GNAT amines,the enzymes amino all group GNAT characterized of theenzymes substrate to datecharacterized [10,31,65,93]. act on primary to Following date amino act the on groups. nucleophilicprimary Generally, amino thegroups. aminoattack Generally, groupon AcCoA of the the a transientamino acceptor group zwitterionic substrate of the needstetrahedacceptor toral besubstrate intermediate deprotonated needs form to to (Figurebe perform deprotonated 22), a the direct breakdown to nucleophilic perform a attackdirectof nucleophilic onwhich the occurs carbonyl attack through carbon on a the proton of carbonyl the transfer enzyme-bound carbon from aof general the AcCoA. enzyme-bound acid (usually Most GNAT Tyr AcCoA. or enzymesSer) Most and mayGNAT have involve an enzymes amino acidhave (usuallydeprotonationan amino Glu, acid Aspof (usuallythe or tetrahedral Ser) Glu, near Asp intermediate their or active Ser) near siteby a that theirgeneral serves active base, as site after a general that which serves basethe acetylatedas that a general extracts product base a proton that fromextractsis the released a amino proton [10,137]. group from He ofthe and the amino substratecolleagues group [114] [10 of,31 thereported,65 ,su93bstrate]. that Following 62% [10,31,65,93]. of the the characterized nucleophilic Following GNAT attack the enzymesnucleophilic on AcCoA aattack transient on AcCoA zwitterionic a transient tetrahedral zwitterionic intermediate tetrahed formral intermediate (Figure 22), form the breakdown (Figure 22), of the which breakdown occurs throughof which a occurs proton through transfer a from proton a general transfer acid from (usually a general Tyr acid or Ser) (usually and may Tyr involveor Ser) deprotonationand may involve of thedeprotonation tetrahedral of intermediate the tetrahedral by a generalintermediate base, afterby a general which the base, acetylated after which product the isacetylated released product [10,137]. Heis released and colleagues [10,137]. [114 He] and reported colleagues that 62% [114] of reported the characterized that 62% GNAT of the enzymescharacterized have GNAT a conserved enzymes Tyr

Int. J. Mol. Sci. 2016, 17, 1018 28 of 45 Int. J. Mol. Sci. 2016, 17, 1018 28 of 44 residuehave a conserved that serves Tyr as aresidue general that acid serves in the as reaction, a general and acid 36% in ofthe them reaction, have aand conserved 36% of them Glu residue have a thatconserved serves Glu as a residue general that base. serves as a general base.

Figure 22. A common general direct acyltransfer mechanism of GNAT superfamily members.

In some GNAT enzymes, two or more amino acid residues together serve to abstract a proton In some GNAT enzymes, two or more amino acid residues together serve to abstract a proton from the amino group of the substrate. Deprotonation of the acceptor substrate by a general base may from the amino group of the substrate. Deprotonation of the acceptor substrate by a general base may also occur via ordered water molecule(s) [23,45,80,132], although some GNAT enzymes do not utilize also occur via ordered water molecule(s) [23,45,80,132], although some GNAT enzymes do not utilize a specific amino acid residue for the substrate deprotonation. For example, the PA4794 protein uses a specific amino acid residue for the substrate deprotonation. For example, the PA4794 protein uses well-ordered water molecules for the deprotonation step [137], while the positive charge around the well-ordered water molecules for the deprotonation step [137], while the positive charge around the entrance to the active site of ScHpa2 is thought to favor a deprotonated state of the substrate, thus entrance to the active site of ScHpa2 is thought to favor a deprotonated state of the substrate, thus negating a requirement for a specific general base residue [76]. Substrates of some GNAT enzymes negating a requirement for a specific general base residue [76]. Substrates of some GNAT enzymes (for (for example, N-acetylglucosamine-6-phosphate, the substrate for ScGNA1) have a lower pKa than example, N-acetylglucosamine-6-phosphate, the substrate for ScGNA1) have a lower pK than that of that of other GNAT acceptor substrates and bind to the enzyme in a deprotonated form,a eliminating other GNAT acceptor substrates and bind to the enzyme in a deprotonated form, eliminating the need the need for a general base [99]. for a general base [99]. Analysis of the pH dependence of the enzymatic activity combined with mutagenesis and Analysis of the pH dependence of the enzymatic activity combined with mutagenesis and structural studies suggested that some members of the GNAT superfamily (e.g., ScGCN5 and structural studies suggested that some members of the GNAT superfamily (e.g., ScGCN5 and SeAAC(6′)-Iy) catalyze the transfer reaction via water—rather than amino acid residue-mediated SeAAC(61)-Iy) catalyze the transfer reaction via water—rather than amino acid residue-mediated protonation of the tetrahedral intermediate; these enzymes appear to have only one catalytic residue protonation of the tetrahedral intermediate; these enzymes appear to have only one catalytic (general base) [39,188]. Interestingly, in contrast to GNAT acetyltransferases, the FemX aminoacyl residue (general base) [39,188]. Interestingly, in contrast to GNAT acetyltransferases, the FemX transferase member of this superfamily follows a substrate-aided transfer mechanism rather than aminoacyl transferase member of this superfamily follows a substrate-aided transfer mechanism using a general catalytic base or acid residing on the protein [158]. The hydrogen bond between the rather than using a general catalytic base or acid residing on the protein [158]. The hydrogen bond carbonyl group of acyl-CoA and the main-chain amide of a residue on the β4 strand is a conserved between the carbonyl group of acyl-CoA and the main-chain amide of a residue on the β4 strand is feature found in many GNAT enzymes. This bond likely stabilizes the tetrahedral reaction a conserved feature found in many GNAT enzymes. This bond likely stabilizes the tetrahedral reaction intermediate [3,10,23,31,68,73,108,147]. intermediate [3,10,23,31,68,73,108,147]. Some GNAT enzymes (for example, hSSAT [132] and (SeAAC(6′)-Iy [22]) follow a random Some GNAT enzymes (for example, hSSAT [132] and (SeAAC(61)-Iy [22]) follow a random kinetic kinetic mechanism, where either AcCoA or an acceptor substrate can bind to the free enzyme first. mechanism, where either AcCoA or an acceptor substrate can bind to the free enzyme first. There There are also examples of GNAT transferases that follow an ordered kinetic mechanism, where are also examples of GNAT transferases that follow an ordered kinetic mechanism, where either either the acceptor substrate binds first followed by AcCoA (e.g., rat liver SSAT [128]) or AcCoA the acceptor substrate binds first followed by AcCoA (e.g., rat liver SSAT [128]) or AcCoA binds binds first, followed by the substrate (e.g., EcAAC(6′)-Ib [25] and OaAANAT [77]. The structural first, followed by the substrate (e.g., EcAAC(61)-Ib [25] and OaAANAT [77]. The structural study of study of OaAANAT revealed that binding of AcCoA to the enzyme induces major structural OaAANAT revealed that binding of AcCoA to the enzyme induces major structural rearrangements rearrangements at its active site that complete the serotonin binding pocket. This provided a at its active site that complete the serotonin binding pocket. This provided a structural basis for the structural basis for the ordered sequential mechanism in OaAANAT.5. Association of Expression of ordered sequential mechanism in OaAANAT. GNAT Enzymes with Cancer 5. AssociationGNAT enzymes of Expression are known of GNATto play Enzymesa role in a with wide Cancer range of human diseases including cancer, diabetes and asthma. Acetylation of histone and non-histone proteins by GNATs controls gene expression,GNAT regulation enzymes areof transcription known toplay factors, a role DNA in replication, a wide range DNA of repair, human cell diseases cycle progression, including cancer,cell signaling diabetes pathways and asthma. and metabolism Acetylation (Table of histone 2) [44,46,189,190], and non-histone and proteinsmisregulation by GNATs of the controlshistone geneN-acetyltransferase expression, regulation (HAT) activity of transcription has been factors,linked with DNA cancer, replication, asthma DNA and repair, viral cellinfections cycle progression,[9,10,189,191,192]. cell signaling A recent pathwaysreview by andKaypee metabolism et al. [189] (Table summarized2)[ 44,46, 189the, 190role], of and aberrant misregulation histone ofacetylation the histone in tumorN-acetyltransferase development and (HAT) discussed activity altered has beenan HAT linked profile with as cancer,a diagnostic asthma tool and for early viral infectionsdetection of[9 ,cancer.10,189, 191Furthermore,,192]. A recent it has review been reported by Kaypee that et a al. substantial [189] summarized increase in the HAT1 role ofexpression aberrant histoneoccurs during acetylation progression in tumor of development liver and colon and cancer discussed [44,193]. altered In an addition, HAT profile abnormal as a diagnostic expression tool of forPCAF early and detection GCN5 has of cancer. been linked Furthermore, to different it has ty beenpes of reported human thatcancers a substantial including increase glioma, inNSCLC HAT1 expression(non-small cell occurs lung during cancer), progression HCC (hepatocellular of liver and carcinoma), colon cancer and [44 colon,,193]. lung, In addition, oral, prostate abnormal and ovarian cancer [189,194,195]. A recent study showed that attenuation of GCN5 expression inhibited

Int. J. Mol. Sci. 2016, 17, 1018 29 of 45 expression of PCAF and GCN5 has been linked to different types of human cancers including glioma, NSCLC (non-small cell lung cancer), HCC (hepatocellular carcinoma), and colon, lung, oral, prostate and ovarian cancer [189,194,195]. A recent study showed that attenuation of GCN5 expression inhibited the growth of colon cancer cells and promoted their apoptosis [195]. PCAF plays an important role in the regulation of activities of several oncogenes and tumor repressors and thereby has a significant effect on cancer development. PCAF can prevent the progression of HCC by inhibiting the serine/threonine protein kinase 1 [189,196]. There is growing evidence that HATs, PCAF and GCN5 can be exploited as anticancer targets. Current knowledge on inhibitors targeting GNAT enzymes for therapeutic use has been reviewed elsewhere [192]. Downregulation of expression of a GNAT enzyme from a different family—arylalkylamine N-acetyltransferase (AANAT)—correlated with development of the biliary cancer, cholangiocarcinoma [197]. Elevated AANAT expression has showed an inhibitory effect on the development of cholangiocarcinoma and increased apoptosis of the cholangiocarcinoma cell line in an in vitro assay. The results of that study suggested the therapeutic importance of AANAT for management of biliary cancer [197]. Altered cellular expression of the polyamine regulatory GNAT enzyme spermidine/spermine N1-acetyltransferase (SSAT) has been linked with a variety human diseases including cancer and rheumatoid arthritis (reviewed in [123,198]). A recent study reported that elevated expression of SSAT is involved in the development of prostate carcinoma and in metastasis [199]. Induction of SSAT by polyamine analogs (for example, BE-3-3-3) resulted in apoptosis of tumor cells which suggested that SSAT can be a potential target for the development of novel therapeutics [123]. Glucosamine-6-phosphate N-acetyltransferase (GNA1) catalyzes the synthesis of the precursor UDP-N-acetylglucosamine (UDP-GlcNAc) in the hexosamine biosynthetic pathway. UDP-GlcNAc serves as a donor substrate for O-GlcNAcylation of a wide variety of cytosolic and nuclear proteins, and thereby plays a significant role in diverse cellular functions including DNA replication, gene transcription, cell growth and metabolism. Abnormal regulation of O-GlcNAcylation has been linked with a number of human diseases including diabetes and breast cancer [200]. Elevated O-GlcNAcylation plays a crucial role in the progression of breast cancer [200,201]. Therefore, GlcNAcylation could be exploited as a novel target for the development of anticancer therapeutics. Aberrant expression/activity of human N-myristoyltransferase (NMT) was observed in colon, stomach, breast, lung, gallbladder and brain cancer (reviewed in [184,202,203]. Inhibiting NMT1 activity resulted in reduced cancer progression in a murine model and increased apoptosis in different cancer cell lines [184,202]. Development of potential therapeutic anticancer drugs targeting human NMTs has been reviewed elsewhere [202].

6. GNAT Enzymes as Potential Targets for Antimicrobial Agents Structural studies on the complexes of microbial GNAT enzymes with their substrates or substrate analogs have provided useful information for the structure-guided design of compounds that can be potentially developed into novel antimicrobial agents. Inhibition of bacterial aminoglycoside N-acetyltransferases (AACs) has been studied extensively because these enzymes modify aminoglycosides that are widely used in clinical settings, thereby conferring bacterial resistance. It was shown that a bisubstrate analog consisting of aminoglycoside covalently linked with CoA inhibited E. coli AAC in an in vitro assay [27]. However, that inhibitor was ineffective in an in vivo assay, likely due to inability to penetrate the bacterial cell wall. Subsequently, it was shown that truncated aminoglycoside-CoA analogs inhibited SeAAC(6)-Iy at micromolar concentrations in vitro [22], and EfAAC(6)-Ii—at nanomolar concentrations, both in vitro and in vivo [36–38], demonstrating that they have potential as lead compounds in the development of novel therapeutics effective against aminoglycoside-resistant bacterial strains expressing AAC(61). Lombes and co-workers [204] used NMR-based approaches to identify non-aminoglycoside-like fragments that showed micromolar-range inhibition of a different type of AAC(61) of E. coli Int. J. Mol. Sci. 2016, 17, 1018 30 of 45

(EcAAC(6)-Ib). Recently, an EcAAC(61)-Ib inhibitor (1-[3-(2-aminoethyl)benzyl]-3-piperidin-1- ylmethyl)pyrrolidin-3-ol), identified by an in silico docking approach [32], was shown to be effective in preventing the growth of an amikacin-resistant A. baumannii clinical strain that expresses AAC(61)-Ib [32]. A different GNAT family that has been targeted in efforts to identify novel antimicrobial compounds is protein N-myristoyltransferases (NMTs). Fungal and parasitic NMTs are essential enzymes that have different substrate specificities compared to their human homologs. In the case of Candida albicans, an opportunistic human fungal pathogen that can cause systemic infections in immunosuppressed patients, benzofuran inhibitors of C. albicans NMT showed fungicidal activity in vivo [205]. Pharmacokinetic study and analysis of structure–activity relationships demonstrated that these inhibitors have high selectivity over human NMT and are therefore good candidates for development into antifungal drugs for human use [205]. Recently, Rackham and colleagues [174,175] identified benzothiophene ring-containing NMT inhibitors that target both blood and liver stage forms of the malaria parasite Plasmodium falciparum. Furthermore, a pyrazole sulfonamides inhibitor of parasitic NMT that shows antimicrobial activity in the initial hemolymphatic stage of human African sleeping sickness caused by Trypanosoma brucei, was identified [181]. Optimization of this compound led to the discovery of T. brucei NMT inhibitors with higher blood-brain barrier permeability and improved selectivity over human NMT, that showed partial efficacy in stage 2 (when the parasite invades the central nervous system) in the mouse model of trypanosomiasis [206]. In addition, T. cruzi NMT was recently validated as a potential target for the development of drugs that target the mammal-dwelling stages of Chagaz disease [207]. It is anticipated that the knowledge of the structural basis behind the reaction and substrate specificity of the enzymes from the GNAT superfamily, gained through the studies of their complexes with inhibitors and substrates, can be exploited for the development of novel therapeutics to combat emerging multidrug-resistant microbial infections. Int. J. Mol. Sci. 2016, 17, 1018 31 of 45

Table 1. General control non-repressible 5-related N-acetyltransferase (GCN5) superfamily members of a known structure (continues on next page).

Family Name EC No. Source Substrates PBD IDs References MtAAC(21)-Ic 2.3.1.- M. tuberculosis aminoglycosides 1M44/1M4D/1M4G/1M4I [23] SmAAC(3)-Ia 2.3.1.81 S. marcescens aminoglycosides 1BO4 [5] EcAAC(61)-Ib 2.3.1.82 E. coli aminoglycosides 1V0C/2BUE/2VQY [25] 1 Aminoglycoside SeAAC(6 )-Ib11 2.3.1.82 S. enterica aminoglycosides 2PR8/2PRB/2QIR [30] N-acetyltransferases SwAAC(61)-Ie 2.3.1.- S. warneri aminoglycosides 4QC6 [208] EfAAC(61)-Ii 2.3.1.82 E. faecium aminoglycosides 1B87/1N71/2A4N [13,21,34] SeAAC(61)-Iy 2.3.1.82 S. enterica aminoglycosides 1S60/1S3Z/1S5K/2VBQ [22,39] MtEis 2.3.1.- M. tuberculosis aminoglycosides 3R1K/3RYO/3SXN/3UY5/4JD6 [19,41,42] hHAT1 2.3.1.48 H. sapiens histone H4 2P0W [45] ScHAT1 2.3.1.48 S. cerevisiae histone H4 1BOB [6] ScEsa1 2.3.1.48 S. cerevisiae histone H4 1MJ9/1MJA/1MJB [57,58] Histone ScGCN5 2.3.1.48 S. cerevisiae histone H3, H4 1YGH [65] N-acetyltransferases TtGCN5 2.3.1.48 T. thermophila histone H3 1QSN/1QSR/1QST/5GCN/1M1D/1PU9/1PUA/1Q2C [67–70] hGCN5 2.3.1.48 H. sapiens histone H3, H4, H2b 1Z4R [66] hPCAF 2.3.1.48 H. sapiens histone H3, H4 1CM0/4NSQ [72,73] ScHpa2 2.3.1.48 S. cerevisiae histone H3, H4 1QSM/1QSO [76] αTAT1 2.3.1.108 H. sapiens α-tubulin 4GS4 [79] Non-histone protein MtPAT 3.4.1.- M. tuberculosis - 4AVA /4AVB/4AVC [81] N-acetyltransferases SsPAT 2.3.1- S. solfataricus - 3F8K [78] hNaa50p 2.3.1.- H. sapiens peptides 3TFY [80] OaAANAT 2.3.1.87 O. aries 2-arylethylamines 1B6B/1CJW/1KUV/1KUX/1KUY/1L0C [77,86,209,210] Arylalkylamine hAANAT 2.3.1.87 H. sapiens 2-arylethylamines 1IB1 [211] N-acetyltransferases DmAANAT 2.3.1.87 D. melanogaster 2-arylethylamines 3TE4 [93] AaAANAT 2.3.1.87 A. aegypti histamine, arylalkylamines, hydrazine 4FD5/4FD4,4FD6/4FD7 [94]

ScGNA1 2.3.1.4 S. cerevisiae D-glucosamine 6-phosphate 1I1D/1I12/1I21 [99] AfGNA1 2.3.1.4 A. fumigatus D-glucosamine 6-phosphate 2VEZ/2VXK [100,212] Glucosamine-6-phosphate hGNA1 2.3.1.4 H. sapiens D-glucosamine 6-phosphate 3CXP/3CXQ/3CXS [101] N-acetyltransferases TbGNA1 2.3.1.4 T. brucei D-glucosamine 6-phosphate 3I3G [213] AtGNA1 2.3.1.4 A. thaliana D-glucosamine 6-phosphate 3T90 [102] CeGNA1 2.3.1.4 C. elegans D-glucosamine 6-phosphate 4AG7/4AG9 [103] MccE EcMccE - E. coli aspartyl-tRNA synthetase 3R95/3R96/3R9E/3R9F [23] Pseudaminic acid HpPseH 2.3.1.202 H. pylori UDP—linked sugar 4RI1 [107] biosynthesis protein H CjPseH 2.3.1.202 C. jejuni UDP—linked sugar 4XPK/4XPL [109]

WecD EcWecD 2.3.1.210 E. coli dTDP-4-amino-4,6-dideoxy-α-D-galactose 2FS5/2FT0 [113] Tabtoxin resistance PsTTR 2.3.1.- P. syringae tabtoxin 1GHE [114] protein

Mpr1 ScMpr1 3.4.1.- S. cerevisiae L-azetidine-2-carboxylic acid 3W6S/3W6X/3W91 [119] Int. J. Mol. Sci. 2016, 17, 1018 32 of 45

Table 1. Cont.

Family Name EC No. Source Substrates PBD IDs References BsPaiA 2.3.1.57 B. subtilis spermidine/spermine 1TIQ [126] TaPaiA 2.3.1.57 T. acidophilum spermidine/spermine 3FIX/3FIX/3NE7/3NE7 [130] Spermidine/spermine hSSAT 2.3.1.57 H. sapiens spermidine/spermine 2BEI/2B3U/2B3V/2B58/2B4B/2B4D/2B5G/2F5I/2G3T/2JEV [129,132,133,214] N1-acetyltransferases MmSSAT 2.3.1.57 M. musculus spermidine/spermine 3BJ7/3BJ8 [128] VcSpeG 2.3.1.57 V. cholerae spermidine/spermine 4NCZ/4JJX/4MHD/4MI4/4R57/4R87 [127] C-terminal Nε-lysine PA4794 2.3.1.- P. aeruginosa C-terminal lysine containing peptide 3PGP/4KOS/4KOT/4KOU/4KOV/4KOW/4KOX/4KOY/4KUA/4L89 [167] protein acetyltransferases MtRv1347c 2.8.3.- M. tuberculosis - 1YK3 [138] SeRimI 2.3.1.128 S. enterica ribosomal protein S18 2CNS/2CNT [147] Ribosomal protein StRimL 2.3.1.- S. typhimurium. ribosomal protein L7/L12 1S7F/1S7K/1S7L/1S7N [108] Nα-acetyltransferases BsYadF - B. subtilis ribosomal protein L12 1NSL [149] Succinyltransferase MtRv0802c 2.8.3.- M. tuberculosis - 2VZY/2VZZ [4] SaFemA 2.3.2.17 S. aureus peptidoglycan precursor, peptides 1LRZ [150] FemABX aminoacyl WvFemX 2.3.2.10 W. viridescens peptidoglycan precursor, peptides 1P4N/1NE9/1XE4/1XF8/1XIX/4II9 [152,158,159] transferases EcLFT 2.3.2.6 E. coli N-terminal Arg/Lys containing proteins 2DPS/2DPT [161] CaNMT 2.3.1.97 C. albicans N-terminal glycyl-peptides 1NMT/1IYK/1IYL [168,215] LdNMT 2.3.1.97 L. donovani N-terminal glycyl-peptides 2WUU [216] 2WSA/3H5Z/4A2Z/4A30/4A31/4A32/4A33/4CGL/4CGM/4CGN/ LmNMT 2.3.1.97 L. major N-terminal glycyl-peptides [178–181] Protein 4CGO/4CGP/4C68/4C7H/4C7I/4CYN/4CYO/4CYP/4CYQ N-myristoyltransferases ScNMT 2.3.1.97 S. cerevisiae N-terminal glycyl-peptides 2NMT/1IIC/1IID/2P6E/2P6F/2P6G [3,169,172] 4B10/4B11/4B12/4B13/4B14/4A95/4BBH/2YNC/2YND/ PvNMT 2.3.1.97 P. vivax N-terminal glycyl-peptides [173–177] 2YNE/4CAE/4CAF TbNMT 2.3.1.97 T. brucei N-terminal glycyl-peptides 2WSA/3H5Z/4A2Z [182] AfNMT 2.3.1.97 A. fumigatus N-terminal glycyl-peptides 4CAX/4CAV/4CAW [183] hNMT 2.3.1.97 H. sapiens N-terminal glycyl-peptides 4C2X/4C2Y/4C2Z [184] Mycothiol synthase MtMshD 2.3.1.189 M. tuberculosis des-acetylmycothiol 1OZP/1P0H/2C27 [186,187] Int. J. Mol. Sci. 2016, 17, 1018 33 of 45

Table 2. Functions of GNAT superfamily members.

Family Functions References Aminoglycoside N-acetyltransferases resistance to aminoglycoside antibiotics [10,19,41,42] histone deposition, transcription activation, chromatin assembly, DNA repair, promoting cancer cell growth Histone N-acetyltransferases [43,44,46,54–56,63] and apoptosis, amino acid biosynthesis in yeast Non-histone protein N-acetyltransferases chromatin regulation, maintaining genome integrity, mRNA and protein stability [78,82,83,90] Arylalkylamine N-acetyltransferases xenobiotic and folate metabolism, biosynthesis of melatonin, sclerotization and neurotransmitter inactivation [91,92,95] biosynthesis of peptidoglycan, chitin, hexosamine and glycophosphatidylinositol; amino sugar and Glucosamine-6-phosphate N-acetyltransferases [10,11,96–98] nucleotide sugar metabolism MccE inactivation of antibiotic microcin C7 [104,105] Pseudaminic acid biosynthesis protein H flagellin glycosylation [106] WecD biosynthesis of enterobacterial common antigen and flagella [110–112] Tabtoxin resistance protein inactivation of tabtoxin and prevention of wildfire disease [117,118]

Mpr1 L-proline and L-arginine metabolism, oxidative stresses resistance and freeze tolerance [119–122] Spermidine/spermine N1-acetyltransferases regulation of cellular polyamine levels and polyamine metabolism [123,124,128] C-terminal Nε-lysine protein acetyltransferases regulation of gene expression [135,136] Ribosomal protein Nα-acetyltransferases regulation of proteins function and stability, inactivation of antibiotic microcin C7 [145–147] Succinyltransferase unknown [4] FemABX aminoacyl transferases peptidoglycan biosynthesis and methicillin resistance [151,153,154] Protein N-myristoyltransferases regulation of protein–protein and protein-cellular membrane interactions, enhancement of protein stability [165,176] biosynthesis of organosulfur compounds, neutralization of electrophiles, regulation of oxidative stress, Mycothiol synthase [10,185] antibiotic resistance and oxidation of formaldehyde Int. J. Mol. Sci. 2016, 17, 1018 34 of 45

7. Conclusions The last two decades have seen a dramatic increase in the amount of structural information available on GNAT enzymes. Although overall amino acid sequence identity among the members of this superfamily is very low, most share common core structural features. GNAT enzymes show significant diversity in their substrate specificities and play an important role in many biological processes. It is anticipated that the knowledge of the structural basis behind the reaction and substrate specificity of these enzymes, gained through studies of their complexes with inhibitors and substrates, can be exploited for the development of novel therapeutics to combat emerging multidrug-resistant microbial infections and other life-threatening human diseases including cancer and metabolic disorders. Future Issues: What roles (allosteric, cooperative, etc.) do different types of oligomerization play in the catalytic activity and specificity of GNAT enzymes? Can structures of GNAT enzymes of unknown function determined by the Structural Biology Consortiums in combination with high-throughput ligand screening be used to predict their putative substrates? How does the three-dimensional structure of a GNAT enzyme define whether it follows a random or an ordered kinetic mechanism? How do GNAT enzymes recognize their partner proteins within the same pathway? Does posttranslational modification of GNAT enzymes occur and how does it affect their structure and function?

Author Contributions: Abu Iftiaf Md Salah Ud-Din and Anna Roujeinikova collected and analyzed data from the available literature; Abu Iftiaf Md Salah Ud-Din, Alexandra Tikhomirova and Anna Roujeinikova wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.

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