The crystal structures of Apo and complexed Saccharomyces cerevisiae GNA1 shed light on the catalytic mechanism of an amino-sugar N-acetyltransferase

The yeast enzymes involved in UDP-GlcNAc biosynthesis are potential targets for antifungal agents. GNA1, a novel member of the Gcn5-related N-acetyltransferase (GNAT) superfamily, participates in UDP-GlcNAc biosynthesis by catalyzing the formation of GlcNAc6P from AcCoA and GlcN6P. We have solved three crystal structures corresponding to the apo Saccharomyces cerevisiae GNA1, the GNA1-AcCoA, and the GNA1-CoA-GlcNAc6P complexes and have refined them to 2.4, 1.3, and 1.8 A resolution, respectively. These structures not only reveal a stable, beta-intertwined, dimeric assembly with the GlcNAc6P binding site located at the dimer interface but also shed light on the catalytic machinery of GNA1 at an atomic level. Hence, they broaden our understanding of structural features required for GNAT activity, provide structural details for related aminoglycoside N-acetyltransferases, and highlight the adaptability of the GNAT superfamily members to acquire various specificities.     

Structural and functional evidence for Bacillus subtilis PaiA as a novel N1-spermidine/spermine acetyltransferase

Bacillus subtilis PaiA has been implicated in the negative control of sporulation as well as production of degradative enzymes. PaiA shares recognizable sequence homology with N-acetyltransferases, including those that can acetylate spermidine/spermine substrates. We have determined the crystal structure of PaiA in complex with CoA at 1.9 A resolution and found that PaiA is a member of the N-acetyltransferase superfamily of enzymes. Unexpectedly, we observed the binding of an oxidized CoA dimer in the active site of PaiA, and the structural information suggests the substrates of the enzyme could be linear, positively charged compounds. Our biochemical characterization is also consistent with this possibility, since purified PaiA possesses N1-acetyltransferase activity toward polyamine substrates including spermidine and spermine. Further, conditional overexpression of PaiA in bacteria results in increased acetylation of endogenous spermidine pools. Thus, our structural and biochemical analyses indicate that PaiA is a novel N-acetyltransferase capable of acetylating both spermidine and spermine. In this way, the pai operon may function in regulating intracellular polyamine concentrations and/or binding capabilities. In addition to preventing toxicity due to polyamine excess, this function may also serve to regulate expression of certain bacterial gene products such as those involved in sporulation.     

Thiolsubtilisin acts as an acetyltransferase in organic solvents

The catalytic mechanism of arylamine N-acetyltransferase has been proposed to involve Cys-His-Asp as its catalytic triad. Thiolsubtilisin, a chemically modified enzyme that has a catalytic triad of Cys-His-Asp at the active site, mimics the catalysis of arylamine N-acetyltransferase, serotonin N-acetyltransferase, histone N-acetyltransferase and amino acid N-acetyltransferase. Thiolsubtilisin not only can catalyze amino acid transacetylation, but is also able to catalyze amine transacetylation. Ethyl acetate was used as the acylating reagent to form N-acetyl amino acids and amines in organic solvents with moderate yield. Hence, these findings broaden our understanding of the structural features required for N-acetyltransferases activity as well as provide a structural relationship between cysteine protease and other N-acyltransferases.     

New N-acetyltransferase fold in the structure and mechanism of the phosphonate biosynthetic enzyme FrbF

The enzyme FrbF from Streptomyces rubellomurinus has attracted significant attention due to its role in the biosynthesis of the antimalarial phosphonate FR-900098. The enzyme catalyzes acetyl transfer onto the hydroxamate of the FR-900098 precursors cytidine 5'-monophosphate-3-aminopropylphosphonate and cytidine 5'-monophosphate-N-hydroxy-3-aminopropylphosphonate. Despite the established function as a bona fide N-acetyltransferase, FrbF shows no sequence similarity to any member of the GCN5-like N-acetyltransferase (GNAT) superfamily. Here, we present the 2.0 ? resolution crystal structure of FrbF in complex with acetyl-CoA, which demonstrates a unique architecture that is distinct from those of canonical GNAT-like acetyltransferases. We also utilized the co-crystal structure to guide structure-function studies that identified the roles of putative active site residues in the acetyltransferase mechanism. The combined biochemical and structural analyses of FrbF provide insights into this previously uncharacterized family of N-acetyltransferases and also provide a molecular framework toward the production of novel N-acyl derivatives of FR-900098.     

Involvement of Cys69 residue in the catalytic mechanism of N-hydroxyarylamine O-acetyltransferase of Salmonella typhimurium. Sequence similarity at the amino acid level suggests a common catalytic mechanism of acetyltransferase for S. typhimurium and higher organisms.

Acetyl-coenzyme A:N-hydroxyarylamine O-acetyltransferase is ubiquitous in species ranging from bacteria to mammals and is involved in the metabolic activation of N-hydroxyarylamines derived from mutagenic and carcinogenic aromatic amines and nitroarenes. The nucleotide sequence of the gene that encodes O-acetyltransferase of Salmonella typhimurium was determined, and its deduced amino acid sequence was compared with those of arylamine N-acetyltransferases (EC 2.3.1.5) of higher organisms. The gene of S. typhimurium encoded a protein with a calculated molecular weight of 32,177. Chromosome DNA of S. typhimurium TA1538/1,8-DNP, an O-acetyltransferase-deficient strain, had a -1 frameshift mutation of CCC to CC at the coding region. To date, 11 genes encoding N-acetyltransferase have been cloned from human, rabbit, hamster, and chicken. The N-terminal region of O-acetyltransferase of S. typhimurium with about 170 amino acids showed 25-33% homology with the corresponding region of N-acetyltransferase of the higher organisms. Of the 5 cysteine residues of O-acetyltransferase of S. typhimurium, Cys69 was the only residue that was conserved in all N-acetyltransferases of the higher organisms. The amino acid sequence of Arg-Gly-Gly-X-Cys, including the Cys69, was highly conserved. The mutant O-acetyltransferase of S. typhimurium, which contained Ala69 instead of Cys69, no longer showed the activities of O- and N-acetyltransferase. These results suggest that the Cys69 of S. typhimurium and its corresponding cysteine residues of the higher organisms are essential for the enzyme activities as acetyl-coenzyme A-binding sites.     

Leishmania major thialysine Nepsilon-acetyltransferase: identification of amino acid residues crucial for substrate binding

Thialysine N(epsilon)-acetyltransferases and spermidine/spermine N-acetyltransferases (SSAT) are closely related members of the GCN5-related N-acetyltransferase superfamily. Accordingly, a putative orthologue from the human protozoan parasite Leishmania major exhibits an almost equal similarity to human SSAT and thialysine N(epsilon)-acetyltransferase. Characterisation of the recombinantly expressed L. major protein indicated that it represents a thialysine N(epsilon)-acetyltransferase, preferring thialysine (S-aminoethyl-l-cysteine) and structurally related amino acids as acceptor molecules. The known thialysine N(epsilon)-acetyltransferases contain five conserved amino acid residues that are replaced in SSAT sequences. Kinetic analyses of the respective recombinant mutant proteins suggest that Ser(82) and Thr(83) of L. major thialysine N(epsilon)-acetyltransferase are key residues for acceptor binding. In addition, the conserved Leu(130) is tentatively involved in specific interaction with the sulphur-containing side chain of thialysine. The presence of these three amino acid residues is suggested to be a means by which thialysine N(epsilon)-acetyltransferases can be distinguished from SSAT sequences.     

The pineal family of aromatic amine N-acetyltransferases

The mammalian pineal gland contains two types of N-acetyltransferases which act on aromatic amines. One type preferentially acetylates arylamines such as phenetidine and aniline, whereas the other preferentially acetylates arylalkylamines such as tryptamine and phenylethylamine. The two enzymes can be distinguished by (1) molecular size, (2) regulation, and (3) inactivation characteristics. Arylalkylamine N-acetyltransferase is involved in the regulation of melatonin synthesis in the pineal gland. A specific function of pineal arylamine N-acetyltransferase has not been established; it may function as a nonspecific detoxification mechanism.     

Characterization of a glucosamine/glucosaminide N-acetyltransferase of Clostridium acetobutylicum

Many bacteria, in particular Gram-positive bacteria, contain high proportions of non-N-acetylated amino sugars, i.e., glucosamine (GlcN) and/or muramic acid, in the peptidoglycan of their cell wall, thereby acquiring resistance to lysozyme. However, muramidases with specificity for non-N-acetylated peptidoglycan have been characterized as part of autolytic systems such as of Clostridium acetobutylicum. We aim to elucidate the recovery pathway for non-N-acetylated peptidoglycan fragments and present here the identification and characterization of an acetyltransferase of novel specificity from C. acetobutylicum, named GlmA (for glucosamine/glucosaminide N-acetyltransferase). The enzyme catalyzes the specific transfer of an acetyl group from acetyl coenzyme A to the primary amino group of GlcN, thereby generating N-acetylglucosamine. GlmA is also able to N-acetylate GlcN residues at the nonreducing end of glycosides such as (partially) non-N-acetylated peptidoglycan fragments and β-1,4-glycosidically linked chitosan oligomers. Km values of 114, 64, and 39 μM were determined for GlcN, (GlcN)2, and (GlcN)3, respectively, and a 3- to 4-fold higher catalytic efficiency was determined for the di- and trisaccharides. GlmA is the first cloned and biochemically characterized glucosamine/glucosaminide N-acetyltransferase and a member of the large GCN5-related N-acetyltransferases (GNAT) superfamily of acetyltransferases. We suggest that GlmA is required for the recovery of non-N-acetylated muropeptides during cell wall rescue in C. acetobutylicum.     

Two arylamine N-acetyltransferases from chicken pineal gland as identified by cDNA cloning

A cDNA library prepared from the poly(A)-rich RNA of the chicken pineal gland obtained at night was screened with the 32P-labeled cDNA of arylamine N-acetyltransferase from the chicken liver recently isolated in this laboratory. Two positive clones (p-NAT-3 and p-NAT-10) that cross-hybridized with the liver cDNA were isolated. The cDNAs did not cross-hybridize each other under a high stringency, indicating that they corresponded to different mRNAs. When the cDNAs were inserted into an expression vector pcDL1 under the control of the early promoter of simian virus 40 and introduced into Chinese hamster ovary cells, both cDNAs expressed arylamine N-acetyltransferase activity in the transfected cells. The nucleotide sequences of the cDNAs were determined, from which amino acid sequences were deduced. Both cDNAs coded for 290 amino acids. Similarities in amino acid sequences were about 60% between p-NAT-3, p-NAT-10 and liver N-acetyltransferases. Poly(A)-rich RNA blot hybridization analysis indicated that p-NAT-3 cDNA detected a 2.2-kb band with the poly(A)-rich RNAs from the brain, gut and, less intensively, spleen, liver and kidney, while p-NAT-10 cDNA hybridized only with the poly(A)-rich RNA from the kidney. Neither cDNA detected any hybridization band with the poly(A)-rich RNA from the pineal gland, suggesting that the contents were low. Genomic Southern blot hybridization analysis showed that p-NAT-3, p-NAT-10 and liver N-acetyltransferases were encoded in a separate single gene. The properties of the enzymes expressed in the transfected cells were compared with N-acetyltransferases from the pineal gland, brain and kidney. On a DEAE-cellulose column, the kidney and p-NAT-10 enzymes appeared in the effluent fraction, whereas the brain and p-NAT-3 enzymes were eluted from the column with a gradient elution at 0.08 M NaCl. The supernatant of the pineal gland obtained in the daytime showed two peaks appearing in the effluent fraction and the eluate fraction at 0.08 M NaCl. The substrate specificity of these enzymes were examined with p-phenetidine, 2-aminofluorene, tryptamine and phenylethylamine as substrates. All the enzymes preferred arylamines to arylalkylamines, indicating that both p-NAT-3 and p-NAT-10 cDNAs encoded arylamine N-acetyltransferases.     

Functional sites and evolutionary connections of acylhomoserine lactone synthases

Acylhomoserine lactone (AHL) synthases act as chemical communication signals or pheromones in Gram-negative bacteria and regulate diverse physiological events in a cell density-dependent manner. The recent crystal structure determination of EsaI, a key enzyme in this pathway, shows that the AHL synthase superfamily members adopt the fold of the N-acetyltransferase superfamily. We suggest, by the identification of intermediate sequences, that the two superfamilies are evolutionarily related. Evolutionary trace analyses of aligned sequences and docking studies have been used to discuss functionally important residues of EsaI homologues.     

X-ray crystallographic studies of serotonin N-acetyltransferase catalysis and inhibition

The structure of serotonin N-acetyltransferase (also known as arylalkylamine N-acetyltransferase; AANAT) bound to a potent bisubstrate analog inhibitor has been determined at 2.0 A resolution using a two-edge (Se, Br) multiwavelength anomalous diffraction (MAD) experiment. This acetyl-CoA dependent enzyme is a member of the GCN5-related family of N-acetyltransferases (GNATs), which share four conserved sequence motifs (A-D). In serotonin N-acetyltransferase, motif A adopts an alpha/beta conformation characteristic of the phylogenetically invariant cofactor binding site seen in all previously characterized GNATs. Motif B displays a significantly lower level of conservation among family members, giving rise to a novel alpha/beta structure for the serotonin binding slot. Utilization of a brominated CoA-S-acetyl-tryptamine-bisubstrate analog inhibitor and the MAD method permitted conclusive identification of two radically different conformations for the tryptamine moiety in the catalytic site (cis and trans). A second high-resolution X-ray structure of the enzyme bound to a bisubstrate analog inhibitor, with a longer tether between the acetyl-CoA and tryptamine moieties, demonstrates only the trans conformation. Given a previous proposal that AANAT can catalyze an alkyltransferase reaction in a conformationally altered active site relative to its acetyltransferase activity, it is possible that the two conformations of the bisubstrate analog observed crystallographically correspond to these alternative reaction pathways. Our findings may ultimately lead to the design of analogs with improved AANAT inhibitory properties for in vivo applications.     

Kinetic and structural analysis of bisubstrate inhibition of the Salmonella enterica aminoglycoside 6'-N-acetyltransferase

Aminoglycosides are antibacterial compounds that act by binding to the A site of the small 30S bacterial ribosomal subunit and inhibiting protein translation. Clinical resistance to aminoglycosides is generally the result of the expression of enzymes that covalently modify the antibiotic, including phosphorylation, adenylylation, and acetylation. Bisubstrate analogs for the aminoglycoside N-acetyltransferases are nanomolar inhibitors of Enterococcus faecium AAC(6')-Ii. However, in the case of the Salmonella enterica aac(6')-Iy-encoded aminoglycoside N-acetyltransferase, we demonstrate that a series of bisubstrate analogs are only micromolar inhibitors. In contrast to studies with AAC(6')-Ii, the inhibition constants toward AAC(6')-Iy are essentially independent of both the identity of the aminoglycoside component of the bisubstrate and the number of carbon atoms that are used to link the CoA and aminoglycoside components. The patterns of inhibition suggest that the CoA portion of the bisubstrate analog can bind to the enzyme-aminoglycoside substrate complex and that the aminoglycoside portion can bind to the enzyme-CoA product complex. However, at the high concentrations of bisubstrate analog used in crystallization experiments, we could crystallize and solve the three-dimensional structure of the enzyme-bisubstrate complex. The structure reveals that both the CoA and aminoglycoside portions bind in essentially the same positions as those previously observed for the enzyme-CoA-ribostamycin complex, with only a modest adjustment to accommodate the "linker". These results are compared to previous studies of the interaction of similar bisubstrate analogs with other aminoglycoside N-acetyltransferases.     

Identification and functional characterization of arylamine N-acetyltransferases in eubacteria: evidence for highly selective acetylation of 5-aminosalicylic acid

Arylamine N-acetyltransferase activity has been described in various bacterial species. Bacterial N-acetyltransferases, including those from bacteria of the gut flora, may be involved in the metabolism of xenobiotics, thereby exerting physiopathological effects. We characterized these enzymes further by steady-state kinetics, time-dependent inhibition, and DNA hybridization in 40 species, mostly from the human intestinal microflora. We report for the first time N-acetyltransferase activity in 11 species of Proteobacteriaceae from seven genera: Citrobacter amalonaticus, Citrobacter farmeri, Citrobacter freundii, Klebsiella ozaenae, Klebsiella oxytoca, Klebsiella rhinoscleromatis, Morganella morganii, Serratia marcescens, Shigella flexneri, Plesiomonas shigelloides, and Vibrio cholerae. We estimated apparent kinetic parameters and found that 5-aminosalicylic acid, a compound efficient in the treatment of inflammatory bowel diseases, was acetylated with a catalytic efficiency 27 to 645 times higher than that for its isomer, 4-aminosalicylic acid. In contrast, para-aminobenzoic acid, a folate precursor in bacteria, was poorly acetylated. Of the wild-type strains studied, Pseudomonas aeruginosa was the best acetylator in terms of both substrate spectrum and catalytic efficiency. DNA hybridization with a Salmonella enterica serovar Typhimurium-derived probe suggested the presence of this enzyme in eight proteobacterial and four gram-positive species. Molecular aspects together with the kinetic data suggest distinct functional features for this class of microbial enzymes.     

Novel bisubstrate analog inhibitors of serotonin N-acetyltransferase: the importance of being neutral

Linker modified novel bisubstrate analog inhibitors 4-7 for serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) have been designed and synthesized. Examination of these inhibitors with AANAT in vitro suggested that: (i) linker hydrogen bonding makes only modest contributions to the affinity of bisubstrate analog inhibitors studied; (ii) greater than or equal to four methylene groups between the indole and the coenzyme A (CoASH) moieties are required for a bisubstrate analog inhibitor to achieve strong AANAT inhibition; (iii) the AANAT active site appears not to accommodate positively charged linkers as well as neutral ones; and (iv) substrate amine pKa depression may constitute one strategy for AANAT substrate recognition and catalysis. The results reported here have enhanced our understanding of AANAT substrate recognition/catalysis, and are important for novel inhibitor design. Since AANAT belongs to the GCN5-related N-acetyltransferase (GNAT) superfamily, our experimental strategies should find applications for other acetyltransferases.     

Purification of the recombinant human serotonin N-acetyltransferase (EC 2.3.1.87): further characterization of and comparison with AANAT from other species

Melatonin is synthesized by a series of enzymes, the penultimate one, serotonin N-acetyltransferase, catalyzing the limiting reaction. In the present study, we compared the recombinant serotonin N-acetyltransferases from rat, ovine, and human. The human protein is particularly difficult to purify because it interacts strongly with a putative chaperone protein from bacteria whereas the rat and sheep enzymes, which interact less strongly with this protein, have been purified close to homogeneity. We identified the contaminating protein as GroEL, the bacterial equivalent of Hsp60. We present numerous catalytic activities (substrate and cosubstrate specificities as well as inhibitor specificities) measured on the three species enzymes from which we deduced that the presence of the chaperone might partly explain the differences between the various species enzyme characteristics, beside the inter-species ones resulting from sequence differences. Despite several trials reported in the literature, a purification to homogeneity of the human (recombinant) enzyme has never been described. We present a new purification method, by using an original denaturation/renaturation process in which the enzyme is immobilized on an affinity chromatography column. The enzyme is then eluted in an active and pure form (i.e., absence of chaperone). The up-scaled system permitted us to perform the necessary experiments for the measurement of more accurate affinities of human serotonin N-acetyltransferase towards its main natural substrates, showing that only the activity of the enzyme towards phenylethylamine was modified.     

MAK3 encodes an N-acetyltransferase whose modification of the L-A gag NH2 terminus is necessary for virus particle assembly.

The MAK3 gene is necessary for propagation of the L-A double-stranded RNA virus of Saccharomyces cerevisiae. MAK3 encodes a protein with substantial homology to the Escherichia coli rimI N-acetyltransferase that acetylates the NH2 terminus of ribosomal protein S18, and shares consensus sequences with a group of N-acetyltransferases. The NH2 terminus of the viral major coat protein encoded by L-A is normally blocked, but we find that it is unblocked in a mak3-1 mutant. L-A virus-encoded proteins produced from a cDNA clone of L-A can encapsidate the L-A (+)-strands in a wild-type host, but not in a mak3-1 mutant strain. The amount of major coat protein found in the particle fraction is reduced greater than 100-fold, and the amount in the total cell extract is reduced 5-10-fold. A modified beta-galactosidase, having as its NH2-terminal the NH2-terminal 13 residues of the L-A-encoded major coat protein, is blocked in a wild-type host, but not in a mak3-1 host. We propose that MAK3 encodes an N-acetyltransferase whose modification of the L-A major coat protein NH2 terminus is essential for viral assembly, and that unassembled coat protein is unstable.     

The structure of arylamine N-acetyltransferase from Mycobacterium smegmatis--an enzyme which inactivates the anti-tubercular drug,isoniazid

Arylamine N-acetyltransferases which acetylate and inactivate isoniazid, an anti-tubercular drug, are found in mycobacteria including Mycobacterium smegmatis and Mycobacterium tuberculosis. We have solved the structure of arylamine N-acetyltransferase from M. smegmatis at a resolution of 1.7 A as a model for the highly homologous NAT from M. tuberculosis. The fold closely resembles that of NAT from Salmonella typhimurium, with a common catalytic triad and domain structure that is similar to certain cysteine proteases. The detailed geometry of the catalytic triad is typical of enzymes which use primary alcohols or thiols as activated nucleophiles. Thermal mobility and structural variations identify parts of NAT which might undergo conformational changes during catalysis. Sequence conservation among eubacterial NATs is restricted to structural residues of the protein core, as well as the active site and a hinge that connects the first two domains of the NAT structure. The structure of M. smegmatis NAT provides a template for modelling the structure of the M. tuberculosis enzyme and for structure-based ligand design as an approach to designing anti-TB drugs.     

Identification of amino acid residues essential for the yeast N-acetyltransferase Mpr1 activity by site-directed mutagenesis

We previously discovered that the budding yeast Saccharomyces cerevisiae Sigma1278b has the MPR1 gene that confers resistance to the proline analogue azetidine-2-carboxylate (AZC). The MPR1-encoded protein (Mpr1) is an N-acetyltransferase that detoxifies AZC and is a novel member of the GCN5-related N-acetyltransferase (GNAT) superfamily. Mpr1 can reduce intracellular oxidation levels and protect yeast cells from oxidative stress, heat shock, freezing, or ethanol treatment. Here, we analyzed the amino acid residues in Mpr1 involved in substrate binding and catalysis by site-directed mutagenesis. The mutated genes were expressed in Escherichia coli, and the recombinant Strep-tagged fusion proteins were analyzed in terms of AZC resistance and acetyltransferase activity. The replacement of Arg145, which is conserved in the GNAT superfamily, by Ala, Asp, Glu, Gly, or Trp led to a growth defect of transformants grown in the presence of AZC. Kinetic studies demonstrated that these mutations caused a large reduction in the affinity for AZC and acetyl-CoA, suggesting that Arg145 interacts with both substrates. Among seven conserved Tyr residues, one of which may be a catalytic residue in the GNAT superfamily, Tyr166Ala- showed no detectable activity and Tyr166Phe-Mpr1, a remarkable decrease of the k(cat)/K(m) value. This result suggests that Tyr166 is critical for the catalysis.     

Serotonin metabolism in rat skin: characterization by liquid chromatography-mass spectrometry

We have recently uncovered the full expression of novel cutaneous serotoninergic and melatoninergic systems in the human and hamster skin. In this work, we have characterized serotonin metabolism in the rat skin using liquid chromatography-mass spectrometry and found that serotonin undergoes acetylation in the presence of acetyl coenzyme A. Inhibition of serotonin acetylation with Cole bisubstrate inhibitor shows that rat skin expresses both arylalkylamine and arylamine N-acetyltransferase activities. The serotonin degradation product-5-hydroxyindole acetic acid is also detected and pargyline (monoaminooxidase inhibitor) suppresses almost completely 5-hydroxyindole acetic acid accumulation. Together with previous data, the present study clearly demonstrates that biotransformation of serotonin in mammalian skin follows two alternate pathways. In the first pathway, serotonin is acetylated by arylalkylamine and arylamine N-acetyltransferases to generate the precursor of melatonin. Alternately, serotonin may undergo oxidative deamination by monoaminooxidase followed by enzymatic degradation by aldehyde dehydrogenase into 5-hydroxyindole acetic acid, which is presumably devoid of biological activity. Thus, the current methodological development of a liquid chromatography-mass spectrometry-based assay allows rapid resolution of the cutaneous metabolism of serotonin.     

Purification and characterization of glutamate N-acetyltransferase involved in citrulline accumulation in wild watermelon

Citrulline is an efficient hydroxyl radical scavenger that can accumulate at concentrations of up to 30 mm in the leaves of wild watermelon during drought in the presence of strong light; however, the mechanism of this accumulation remains unclear. In this study, we characterized wild watermelon glutamate N-acetyltransferase (CLGAT) that catalyses the transacetylation reaction between acetylornithine and glutamate to form acetylglutamate and ornithine, thereby functioning in the first and fifth steps in citrulline biosynthesis. CLGAT enzyme purified 7000-fold from leaves was composed of two subunits with different N-terminal amino acid sequences. Analysis of the corresponding cDNA revealed that these two subunits have molecular masses of 21.3 and 23.5 kDa and are derived from a single precursor polypeptide, suggesting that the CLGAT precursor is cleaved autocatalytically at the conserved ATML motif, as in other glutamate N-acetyltransferases of microorganisms. A green fluorescence protein assay revealed that the first 26-amino acid sequence at the N-terminus of the precursor functions as a chloroplast transit peptide. The CLGAT exhibited thermostability up to 70 degrees C, suggesting an increase in enzyme activity under high leaf temperature conditions during drought/strong-light stresses. Moreover, CLGAT was not inhibited by citrulline or arginine at physiologically relevant high concentrations. These findings suggest that CLGAT can effectively participate in the biosynthesis of citrulline in wild watermelon leaves during drought/strong-light stress.