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.     

Reversible inhibition of the human xenobiotic-metabolizing enzyme arylamine N-acetyltransferase 1 by S-nitrosothiols

Human arylamine N-acetyltransferase 1 (NAT1) is a polymorphic phase II xenobiotic-metabolizing enzyme which catalyzes the biotransformation of primary aromatic amines, hydrazine drugs, and carcinogens. Structural and functional studies have shown that the NAT1 and factor XIII transglutaminase catalytic pockets are structurally related with the existence of a conserved catalytic triad (Cys-His-Asp). In addition, it has been reported that factor XIII transglutaminase activity could be regulated by nitric oxide (NO), in particular S-nitrosothiols (RSNO). We thus tested whether NAT1 could be a target of S-nitrosothiols. We show here that human NAT1 is reversibly inactivated by S-nitrosothiols such as SNAP (S-nitroso-N-acetyl-DL-penicillamine). A second-order rate constant for the inactivation of NAT1 by SNAP was determined (k(inact)=270M(-1)min(-1)) and shown to be in the same range of values reported for other enzymes. The inhibition of NAT1 by S-nitrosothiols was reversed by dithiothreitol and reduced glutathione, but not by ascorbate. As reported for some reactive cysteine-containing enzymes, our results suggest that inactivation of NAT1 by S-nitrosothiols is due to direct attack of the highly reactive cysteine residue in the enzyme active site on the sulfur of S-nitrosothiols to form a mixed disulfide between these NO-derived oxidants and NAT1. Finally, our findings suggest that, in addition to the polymorphic-dependent variation of NAT1 activity, NO-derived oxidants, in particular S-nitrosothiols, could also regulate NAT1 activity.     

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.     

Crystal structure of tabtoxin resistance protein complexed with acetyl coenzyme A reveals the mechanism for beta-lactam acetylation

Tabtoxin resistance protein (TTR) is an enzyme that renders tabtoxin-producing pathogens, such as Pseudomonas syringae, tolerant to their own phytotoxins. Here, we report the crystal structure of TTR complexed with its natural cofactor, acetyl coenzyme A (AcCoA), to 1.55A resolution. The binary complex forms a characteristic "V" shape for substrate binding and contains the four motifs conserved in the GCN5-related N-acetyltransferase (GNAT) superfamily, which also includes the histone acetyltransferases (HATs). A single-step mechanism is proposed to explain the function of three conserved residues, Glu92, Asp130 and Tyr141, in catalyzing the acetyl group transfer to its substrate. We also report that TTR possesses HAT activity and suggest an evolutionary relationship between TTR and other GNAT members.     

Acceptor substrate binding revealed by crystal structure of human glucosamine-6-phosphate N-acetyltransferase 1

Glucosamine-6-phosphate (GlcN6P) N-acetyltransferase 1 (GNA1) is a key enzyme in the pathway toward biosynthesis of UDP-N-acetylglucosamine, an important donor substrate for N-linked glycosylation. GNA1 catalyzes the formation of N-acetylglucosamine-6-phosphate (GlcNAc6P) from acetyl-CoA (AcCoA) and the acceptor substrate GlcN6P. Here, we report crystal structures of human GNA1, including apo GNA1, the GNA1-GlcN6P complex and an E156A mutant. Our work showed that GlcN6P binds to GNA1 without the help of AcCoA binding. Structural analyses and mutagenesis studies have shed lights on the charge distribution in the GlcN6P binding pocket, and an important role for Glu156 in the substrate binding. Hence, these findings have broadened our knowledge of structural features required for the substrate affinity of GNA1. STRUCTURED SUMMARY:     

Binding of the anti-tubercular drug isoniazid to the arylamine N-acetyltransferase protein from Mycobacterium smegmatis

Isoniazid is a frontline drug used in the treatment of tuberculosis (TB). Isoniazid is a prodrug, requiring activation in the mycobacterial cell by the catalase/peroxidase activity of the katG gene product. TB kills two million people every year and the situation is getting worse due to the increase in prevalence of HIV/AIDS and emergence of multidrug-resistant strains of TB. Arylamine N-acetyltransferase (NAT) is a drug-metabolizing enzyme (E.C. 2.1.3.5). NAT can acetylate isoniazid, transferring an acetyl group from acetyl coenzyme A onto the terminal nitrogen of the drug, which in its N-acetylated form is therapeutically inactive. The bacterium responsible for TB, Mycobacterium tuberculosis, contains and expresses the gene encoding the NAT protein. Isoniazid binds to the NAT protein from Salmonella typhimurium and we report here the mode of binding of isoniazid in the NAT enzyme from Mycobacterium smegmatis, closely related to the M. tuberculosis and S. typhimurium NAT enzymes. The mode of binding of isoniazid to M. smegmatis NAT has been determined using data collected from two distinct crystal forms. We can say with confidence that the observed mode of binding of isoniazid is not an artifact of the crystallization conditions used. The NAT enzyme is active in mycobacterial cells and we propose that isoniazid binds to the NAT enzyme in these cells. NAT activity in M. tuberculosis is likely therefore to modulate the degree of activation of isoniazid by other enzymes within the mycobacterial cell. The structure of NAT with isoniazid bound will facilitate rational drug design for anti-tubercular therapy.     

Structural analysis of a putative aminoglycoside N-acetyltransferase from Bacillus anthracis

For the last decade, worldwide efforts for the treatment of anthrax infection have focused on developing effective vaccines. Patients that are already infected are still treated traditionally using different types of standard antimicrobial agents. The most popular are antibiotics such as tetracyclines and fluoroquinolones. While aminoglycosides appear to be less effective antimicrobial agents than other antibiotics, synthetic aminoglycosides have been shown to act as potent inhibitors of anthrax lethal factor and may have potential application as antitoxins. Here, we present a structural analysis of the BA2930 protein, a putative aminoglycoside acetyltransferase, which may be a component of the bacterium's aminoglycoside resistance mechanism. The determined structures revealed details of a fold characteristic only for one other protein structure in the Protein Data Bank, namely, YokD from Bacillus subtilis. Both BA2930 and YokD are members of the Antibiotic_NAT superfamily (PF02522). Sequential and structural analyses showed that residues conserved throughout the Antibiotic_NAT superfamily are responsible for the binding of the cofactor acetyl coenzyme A. The interaction of BA2930 with cofactors was characterized by both crystallographic and binding studies.     

Insights into how protein dynamics affects arylamine N-acetyltransferase catalysis

Arylamine N-acetyltransferases (NATs) detoxify arylamines and hydrazine xenobiotics by catalyzing their N-acetylation, which prevents their bioactivation. Here, we reveal how structural dynamics impact NAT protein function. Our data suggest that there are multiple conformations in the catalytic cavity of hamster NAT2 that exchange on the millisecond time scale and enable NATs to accommodate substrates of varying size. The regions spanning N177-L180 and D285-F288, which form unique structures in mammalian NATs, possess inherent motions on the nanosecond time scale. The latter segment becomes more restricted in its motions upon substrate binding according to our NMR XNOE data. This greater rigidity appears to stem from interactions with the substrate. Finally, NAT acetylation has been suggested to protect these enzymes from ubiquitination. Our NMR data on a catalytically active state of hamster NAT2 suggest that structural rearrangements caused by its acetylation might contribute to this protection.     

X-ray crystallographic and high-resolution NMR spectroscopy characterization of 4,6-di-O-acetyl-2,3-dideoxy-alpha-D-erythro-hex-2-enopyranosyl sulfamide

Single crystal X-ray diffraction and high-resolution ¹H and ¹³C NMR spectral data for 4,6-di-O-acetyl-2,3-dideoxy-α-d-erythro-hex-2-enopyranosyl sulfamide, a selective inhibitor of carbonic anhydrase isozyme IX, are reported. The ⁰H₅ was found to be the preferred form for this glycosyl sulfamide, both in the crystal lattice and in solution.     

Arylalkylamine N-acetyltransferase gene expression in retina and pineal gland of rats under various photoperiods

The present study examines how the circadian oscillators in the retina and the suprachiasmatic nucleus (SCN) respond to changes in photoperiod. Arylalkylamine N-acetyltransferase (aa-nat) gene expression studied by quantitative RT-PCR revealed that in adult Sprague-Dawley rats kept under different light-dark (LD) cycles for two weeks the temporal pattern of AA-NAT mRNA expression was identical in retina and pineal gland. In both tissues, the time span between the onset of darkness and the nocturnal rise in AA-NAT mRNA expression was 3 h under LD 20:4, 6 h under LD 12:12, and 15 h under LD 4:20. As aa-nat expression in the pineal gland is regulated by the circadian oscillator in SCN, the results suggest that the photoperiodic differences accompanying the seasons of the year are imprinted in more than one oscillator and that this may accentuate the important message regarding 'time of year.'     

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.     

A density functional theory study on the role of His-107 in arylamine N-acetyltransferase 2 acetylation

Arylamine N-acetyltransferases (NATs, EC 2.3.1.5) catalyze an acetyl group transfer from acetyl coenzyme A (AcCoA) to primary arylamines, and are responsible for the biotransformation and metabolism of drugs, carcinogens, etc. Structure analysis revealed that His-107 was likely the residue accountable for mediating acetyl transfer. We have examined the full catalytic mechanism of this system by means of DFT method. The results indicate that if the acetyl group directly transferred from the donor, p-nitrophenyl acetate, to the acceptor, cysteine, the high activation energy will be a great hindrance. These energies have dropped a little in a range of 20-25 kJ/mol when His-107 is assisting the transfer process. However, when protonated His-107 is mediating the reaction, the activation energies have dropped about 70-85 kJ/mol. Our calculations strongly support an enzymatic acetylation mechanism that experiences a thiolate-imidazolium pair, which have verified the presumption from experiments.     

Continuous spectrophotometric assays for three regulatory enzymes of the arginine biosynthetic pathway

N-Acetylglutamate synthase (AGS), N-acetylglutamate kinase (AGK), and glutamate N-acetyltransferase (GAT) are the key enzymes in the synthesis of arginine that serves as an important precursor for the synthesis of protein, polyamines, urea, and nitric oxide. Current assays available for these three enzymes are laborious and time-consuming and do not allow continuous monitoring of enzyme activities. Here we established continuous enzyme assays for AGS, AGK, and GAT based on the coupling of AGS and GAT reactions to AGK followed by coupling of the AGK reaction to N-acetylglutamate 5-phosphate reductase (AGPR). The rate of AGPR-dependent oxidation of reduced nicotinamide adenine dinucleotide phosphate was monitored continuously as a change in absorbance at 340 nm using spectrophotometry. These methods were applied to kinetic analyses for Escherichia coli AGK, E. coli AGS, and Saccharomyces cerevisiae GAT, and the kinetic parameters obtained in the coupling assays showed nearly the same values as those obtained previously using discontinuous assays. The specificity of these coupled assays was confirmed by the lack of enzyme activity from extracts of E. coli AGS-, E. coli AGK-, and S. cerevisiae GAT-deletion mutants. Moreover, the coupled assay enabled us to measure AGS activity from mammalian liver mitochondrial extracts, known to be an important regulatory enzyme for the urea cycle. These coupled enzyme assays are rapid, highly sensitive, and reproducible.     

Arylamine N-acetyltransferase aggregation and constitutive ubiquitylation

Arylamine N-acetyltransferases (NAT1 and NAT2) acetylate and detoxify arylamine carcinogens. Humans harboring certain genetic variations within the NAT genes exhibit increased likelihood of developing various cancer types, especially urinary bladder cancer. Such DNA polymorphisms result in protein products with reduced cellular activity, which is proposed to be due to their constitutive ubiquitylation and enhanced proteasomal degradation. To identify the properties that lead to the reduced cellular activity of certain NAT variants, we introduced one such polymorphism into the human NAT1 ortholog hamster NAT2. The polymorphism chosen was human NAT1*17, which results in the replacement of R64 with a tryptophan residue, and we demonstrate this substitution to cause hamster NAT2 to be constitutively ubiquitylated. Biophysical characterization of the hamster NAT2 R64W variant revealed that its overall protein structure and thermostability are not compromised. In addition, we used steady-state kinetics experiments to demonstrate that the R64W mutation does not interfere with NAT catalysis in vitro. Hence, the constitutive ubiquitylation of this variant is not caused by its inability to be acetylated. Instead, we demonstrate this mutation to cause the hamster NAT2 protein to aggregate in vitro and in vivo. Importantly, we tested and confirmed that the R64W mutation also causes human NAT1 to aggregate in cultured cells. By using homology modeling, we demonstrate that R64 is located at a peripheral location, which provides an explanation for how the NAT protein structure is not significantly disturbed by its mutation to tryptophan. Altogether, we provide fundamental information on why humans harboring certain NAT variants exhibit reduced acetylation capabilities.     

Insight into the structure of Mesorhizobium loti arylamine N-acetyltransferase 2 (MLNAT2): a biochemical and computational study

The arylamine N-acetyltransferases (NAT; EC 2.3.1.5) are xenobiotic-metabolizing enzymes (XME) that catalyze the transfer of an acetyl group from acetylCoA (Ac-CoA) to arylamine, hydrazines and their N-hydroxylated metabolites. Eukaryotes may have up to three NAT isoforms, but Mesorhizobium loti is the only prokaryote with two functional NAT isoforms (MLNAT1 and MLNAT2). The three-dimensional structure of MLNAT1 has been determined (Holton, S.J., Dairou, J., Sandy, J., Rodrigues-Lima, F., Dupret, J.M., Noble, M.E.M. and Sim, E. (2005) Structure of Mesorhizobium loti arylamine N-acetyltransferase 1. Acta Cryst, F61, 14-16). No MLNAT2 crystals have yet been produced, despite the production of sufficient quantities of pure protein. Using purified recombinant MLNAT1 and MLNAT2, we showed here that MLNAT1 was intrinsically more stable than MLNAT2. To test whether different structural features could explain these differences in intrinsic stability, we constructed a high-quality homology model for MLNAT2 based on far UV-CD data. Despite low levels of sequence identity with other prokaryotic NAT enzymes ( approximately 28% identity), this model suggests that MLNAT2 adopts the characteristic three-domain NAT fold. More importantly, molecular dynamics simulations on the structures of MLNAT1 and MLNAT2 suggested that MLNAT2 was less stable than MLNAT1 due to differences in amino-acid sequence/structure features in the alpha/beta lid domain.     

Site-directed mutagenesis studies of acetylglutamate synthase delineate the site for the arginine inhibitor

N-acetyl-l-glutamate synthase (NAGS), the first enzyme of bacterial/plant arginine biosynthesis and an essential activator of the urea cycle in animals, is, respectively, arginine-inhibited and activated. Site-directed mutagenesis of recombinant Pseudomonas aeruginosa NAGS (PaNAGS) delineates the arginine site in the PaNAGS acetylglutamate kinase-like domain, and, by extension, in human NAGS. Key residues for glutamate binding are identified in the acetyltransferase domain. However, the acetylglutamate kinase-like domain may modulate glutamate binding, since one mutation affecting this domain increases the K_m for glutamate. The effects on PaNAGS of two mutations found in human NAGS deficiency support the similarity of bacterial and human NAGSs despite their low sequence identity.     

Theoretical evaluation and improvement on the potency of the rhodanine-based inhibitors for human serotonin N-acetyltransferase

Human serotonin N-acetyltransferase (hAANAT), included in the melatonin biosynthesis, plays a pivotal role in the regulation of the biological clock and the daily rhythm. In this research, a reliable model of hAANAT was first constructed by the homology modelling method. Then the inhibition mode of two representative rhodanine-based inhibitors was explored by molecular dynamics simulations and energy analyses. The results show that the inhibitor class could share a similar inhibition mechanism in which the carboxyl moiety is positioned in the Ac-CoA binding region while the other end spans the serotonin binding pocket. The interaction between the inhibitor's carboxyl and the enzyme seems to be more important according to the decomposition of binding free energy. Based on the proposed inhibition mode, the inhibitor's improvement was carried out to obtain a more potent compound. The newly designed inhibitor, with the larger binding free energy, exhibits the stronger interaction with the related residues of the enzyme by the added chemical groups. This work will shed light on the inhibition mechanism of the rhodanine-based inhibitors and promote the development of a new drug targeting hAANAT.     

On the structural significance of the linkage region constituents of N-glycoproteins: an X-ray crystallographic investigation using models and analogs

The linkage region constituents, namely, 2-acetamido-2-deoxy-beta-D-glucopyranose and asparagine are conserved in the N-glycoproteins of all the eukaryotes. The present work is aimed at understanding the reasons for the occurrence of GlcNAc and Asn as the linkage region constituents. A total of six sugar amides have been designed as models and analogs of the linkage region and their crystal structures have been solved. This is the first report on the X-ray crystallographic investigation of the effect of systematic changes in the linkage sugar as well as its aglycon moiety on the N-glycosidic torsion, psi(N) (O5-C1-N1-C1(')). This also forms the first report on the crystal structure of a model of L-RhabetaAsn, a variant linkage found in the surface layer glycoprotein of Bacillus stearothermophillus. Among the models and analogs examined, the acetamido derivatives of Man and Xyl, the linkage sugars of O-glycoproteins, show a psi(N) value of -114.5 degrees and -121.2 degrees, respectively, deviating maximum from the value of -89.8 degrees reported for the model compound GlcNAcbetaNHAc. The L-Rha and Gal derivatives also show noticeable deviations. The psi(N) values, -89.5 degrees and -91.0 degrees, of the propionamide derivatives of Glc and GlcNAc (analogs of GlcbetaGln and GlcNAcbetaGln, respectively) agree well with those (-93.8 degrees and -89.8 degrees ) reported for their corresponding acetamide derivatives suggesting Gln could serve as well as Asn as the linkage region amino acid. However, the rotational freedom about the additional C-C bond would lead to altered rigidity of the linkage region. An analysis of packing reveals that the molecular assembly of these compounds is driven by different infinite and finite chains of hydrogen bonds. The double pillaring of hydrogen bonds involving the amide groups at C1 and C2 is seen as a unique packing feature characteristic of beta-1-N-acyl derivatives of GlcNAc. Based on the findings of the present study, it is speculated that the linkage region constituents of the eukaryotic N-glycoproteins appear to fulfill three essential structural requirements: rigidity, planarity, and linearity and these are met by the trisaccharide core and Asn at the linkage region.     

Ex vivo studies on the acute and chronic effects of DHEA and DHEA-sulfate on melatonin synthesis in young- and old-rat pineal glands

This study investigates the effects of acute and chronic injections of the neurosteroid dehydroepiandrosterone (DHEA) and its sulfate DHEA-S on pineal gland melatonin synthesis. Pineal melatonin production and plasma melatonin levels were investigated in young (9-week-old) and old (27-month-old) male Wistar rats. DHEA or DHEA-S have been administered acutely in a single intraperitoneal injection at a dosage of 50, 250, or 500 microg per animal, or on a long-term basis, i.e., for 8 days at a dosage of 100 microg per animal, 1 h before the onset of darkness. DHEA, at a dose of 50, 250, or 500 microg per animal, administered acutely to rats had no significant effects on pineal melatonin production whatever the age of the animals. In contrast, 500 microg DHEA-S induced a significant increase in the pineal melatonin content (15% in young animals and 35% in old animals) and the activity of N-acetyltransferase, the rate-limiting enzyme for melatonin synthesis in the pineal gland, (40% in young animals and 20% in old animals), without altering the activity of hydroxyindole-O-methyltransferase whatever the age of the animals. At lower concentrations (50 or 250 microg) DHEA-S had no effect on pineal melatonin production regardless of the age of the rats. Chronic injection of DHEA or DHEA-S at a dose of 100 microg had no effect on pineal melatonin or NAT and HIOMT activities in the two age groups. This work shows that DHEA-S (and not DHEA) is able, at pharmacological concentrations, to stimulate melatonin production by rat pineal glands regardless of the age of the animals.     

Assay of the pyruvate dehydrogenase complex by coupling with recombinant chicken liver arylamine N-acetyltransferase

The activity of the pyruvate dehydrogenase complex has long been determined in some laboratories by coupling the production of acetyl-coenzyme A (acetyl-CoA) to the acetylation of 4-aminoazobenzene-4'-sulfonic acid by arylamine N-acetyltransferase. The assay has some advantages, but its use has been limited by the need for large amounts of arylamine N-acetyltransferase. Here we report production of recombinant chicken liver arylamine N-acetyltransferase and optimization of its use in miniaturized assays for the pyruvate dehydrogenase complex and its kinase.