- Research article
- Open Access
Kinetic characterisation of arylamine N-acetyltransferase from Pseudomonas aeruginosa
© Westwood and Sim; licensee BioMed Central Ltd. 2007
- Received: 14 November 2006
- Accepted: 20 March 2007
- Published: 20 March 2007
Arylamine N-acetyltransferases (NATs) are important drug- and carcinogen-metabolising enzymes that catalyse the transfer of an acetyl group from a donor, such as acetyl coenzyme A, to an aromatic or heterocyclic amine, hydrazine, hydrazide or N-hydroxylamine acceptor substrate. NATs are found in eukaryotes and prokaryotes, and they may also have an endogenous function in addition to drug metabolism. For example, NAT from Mycobacterium tuberculosis has been proposed to have a role in cell wall lipid biosynthesis, and is therefore of interest as a potential drug target. To date there have been no studies investigating the kinetic mechanism of a bacterial NAT enzyme.
We have determined that NAT from Pseudomonas aeruginosa, which has been described as a model for NAT from M. tuberculosis, follows a Ping Pong Bi Bi kinetic mechanism. We also describe substrate inhibition by 5-aminosalicylic acid, in which the substrate binds both to the free form of the enzyme and the acetyl coenzyme A-enzyme complex in non-productive reaction pathways. The true kinetic parameters for the NAT-catalysed acetylation of 5-aminosalicylic acid with acetyl coenzyme A as the co-factor have been established, validating earlier approximations.
This is the first reported study investigating the kinetic mechanism of a bacterial NAT enzyme. Additionally, the methods used herein can be applied to investigations of the interactions of NAT enzymes with new chemical entities which are NAT ligands. This is likely to be useful in the design of novel potential anti-tubercular agents.
- Acceptor Substrate
- Acetyl Coenzyme
- Enzyme Intermediate
- Ping Pong Mechanism
- Pigeon Liver
Arylamine N-acetyltransferases (NATs, E.C. 220.127.116.11) are a family of enzymes (30–34 kDa) found in a range of eukaryotes and prokaryotes. NATs catalyse the transfer of an acetyl group from a donor, such as acetyl coenzyme A (AcCoA) to an aromatic or heterocyclic amine, hydrazine, hydrazide or N-hydroxylamine acceptor substrate.
The NAT enzymes in prokaryotes, particularly NAT from S. typhimurium , have been important in studies of the metabolism of carcinogens. Recent evidence suggests that prokaryotic NATs may also have an endogenous role. For example, a NAT-like protein in Amycolatopsis mediterranei (RifF) is responsible for the final ring-closure step in the biosynthesis of the rifamycin precursor, proansamycin X . Although the precise endogenous function of NAT in mycobacteria has not been established, genetic studies suggest strongly that NAT has a role in cell wall complex lipid biosynthesis in Mycobacterium bovis BCG . It has been proposed that NAT represents a good anti-tubercular target, since ablation of the nat gene results in increased intracellular killing of mycobacteria within macrophage . The NAT from Mycobacterium tuberculosis has not been expressed as a soluble recombinant enzyme in sufficient quantities for detailed activity studies, although other bacterial NATs are available. These include NATs from Salmonella typhimurium , Mycobacterium smegmatis , Mesorhizobium loti  and Pseudomonas aeruginosa . The three-dimensional structures of these enzymes have been solved by X-ray crystallography. The overall fold is superimposable [6–9], and all four enzymes share a catalytic triad of residues: Cys-His-Asp , a motif which is completely conserved throughout all known active NATs .
As a start to the process of identifying novel NAT ligands, Brooke and others have developed a method suitable for the rapid identification of NAT substrates and inhibitors with the colorimetric agent 5,5'-dithio-bis(2-nitrobenzoic acid) (Ellman's reagent, DTNB) . The extent of reaction is measured by detecting the coloured 5-thio-2-nitrobenzoic acid, produced by reaction of DTNB with the free thiol CoA, formed during the NAT reaction [11, 12]. Brooke and co-workers have used this method to identify novel inhibitors of bacterial NATs with the aim of developing novel antimycobacterial agents . While the structures of bacterial NATs are known, to date, there have been no kinetic analyses of prokaryotic NAT enzymes and it is not known whether these enzymes share the kinetic mechanism previously determined for their eukaryotic counterparts.
The eukaryotic NAT enzymes have been investigated in relation to their role in drug- and carcinogen-metabolism [14–16], and it has also been suggested that certain eukaryotic NATs have an endogenous role . The crystal structure of human NAT1 (F125S mutant, pdb code 2IJA) has recently been solved. This crystal structure, along with an NMR-derived model of human NAT1  and homology models of human NAT1 , human NAT2  and hamster NAT2 , all show that the three-dimensional fold is very highly conserved throughout the NAT family, with very similar positioning of the catalytic triad residues. The most notable difference between eukaryotic and prokaryotic NATs is the existence of a loop region between the second and third domains in eukaryotic NATs, which is unlikely to have a role in catalysis [21, 22].
The determination of kinetic constants for NAT, in particular with AcCoA as the acetyl donor, is experimentally challenging [23, 24]. The apparent Michaelis constants and limiting rates of N-acetylation are dependent on the concentrations of the acetyl donor and acceptor. There are several features of the reactants which limit the determination of kinetic parameters by linear methods, including limited solubility and very high optical absorbance. Despite the technical difficulties associated with NAT enzyme assays, the enzymatic reactions of NATs from pigeon liver  and rabbit liver  preparations have been shown to follow the Ping Pong Bi Bi kinetic mechanism . Approximate kinetic parameters have been determined for several eukaryotic NAT enzymes [23, 25, 26, 28–33], and Wagner, Hanna and colleagues have recently determined the catalytic mechanism of pure recombinant NAT2 from Syrian hamsters [34, 35]. No such analysis has been carried out so far for any of the bacterial NATs.
The NAT enzyme from P. aeruginosa (PANAT) has been cloned, expressed, characterised and crystallised , and is an ideal enzyme system for the study of the kinetics of a bacterial member of this unique enzyme family. The PANAT enzyme is very stable and highly active relative to other bacterial NATs currently available as pure proteins [4, 5, 7].
Determination of the kinetic mechanism and true kinetic parameters by normalised plot analysis
We have used a normalised plot method, described by Bravo and colleagues , to elucidate the kinetic mechanism of the PANAT-catalysed N-acetylation of 5-aminosalicylic acid with AcCoA as acetyl donor, and to measure the true kinetic parameters for the substrates.
A flowchart describing the normalised plot method is presented as supplemental data [see Additional file 1]. In this method, the normalised concentration (A') is defined as an arbitrary constant concentration (A) multiplied by a numerical factor (a) as shown in equation 1.
A' = aA (1)
The initial rates of reaction are measured in three separate experimental series. For the series b = 1, reaction velocities (Va,b) are measured in the presence of a constant concentration of 5-aminosalicylic acid (B), and a series of actual concentrations of AcCoA which are described as multiples of the arbitrarily fixed concentration A (e.g. for a concentration of , a = 0.25). Similarly, for the series a = 1, Va,bvalues are measured in the presence of AcCoA (at concentration A) and a series of concentrations of 5-aminosalicylic acid (multiples of B). For the final series of data (a = b), the concentrations of substrates are varied in identical proportions (e.g. and , and ). The experiment is designed such that the initial reaction velocity where a = b = 1 (V1,1, eq. 2) is measured in each series of collected data. The normalised velocity, (eq. 2), is a corrected reciprocal value of the experimentally determined value.
Definitions of parameters in the normalised velocity equation (eq 3)a
K iB K mA
Appears in Ordered Bi Bi only
Accounts for substrate inhibition by 5-aminosalicylic acid on the free enzyme
Accounts for substrate inhibition by 5-aminosalicylic acid on the enzyme·AcCoA complex
The denominator term den is independent of both a and b
Results of non-linear regression by the least-squares method.a
α = 0
α = β = 0
β = 0
0.219 ± 0.073**
0.199 ± 0.174†
0.219 ± 0.120†
0.199 ± 0.279†
1.02 ± 0.29**
1.51 ± 1.03†
1.02 ± 0.46*
1.51 ± 1.53†
1.93 ± 0.28***
1.93 ± 0.37***
1.03 ± 0.34**
0.550 ± 0.403†
1.03 ± 0.50†
0.550 ± 0.560†
~0.00 ± 0.01†
~0.00 ± 0.01†
0.422 ± 0.105***
0.512 ± 0.327†
0.422 ± 0.156*
0.512 ± 0.463†
Sum of squares
Bi Bi Mechanism
Substrate inhibition by 5-aminosalicylic acid on free enzyme?
The Ordered Bi Bi term α was found to reduce to zero when included in the calculation, hence the numbers generated with equations derived for mechanisms 3O and 4O were the same as those from mechanisms 1P and 2P respectively. In all cases, the den and den(calc) values were very similar; however, the sum of squares from the calculations where β was set to 0 were over four-fold higher than those where β was included. The statistical significance of each solution (determined with Student's t test within KyPlot), indicates that mechanism 1P is the most likely. In order to determine by independent means whether mechanism 1P or 2P is most appropriate, the method described by Mannervik was used , in which the quotient, Fexp (eq. 4) is compared with the F-statistic: F(pk - pj, n - pk), where SSj (= 1.254) is the residual sum of squares from the simpler model (mechanism 2P) with pj (= 4) parameters, SSk (= 0.268) is the residual sum of squares from the more complex model (mechanism 1P) with pk (= 5) parameters and n (= 27) is the number of data points.
Thus, from the four rival kinetic mechanisms considered (Table 2), an Ordered mechanism can be ruled out, based on the reduction of the denominator term α to zero in the calculation process, the correlations of the rival kinetic models and the statistical significance of the fits. Of the two remaining Ping Pong mechanisms, mechanism 2P is a poorer fit than mechanism 1P, as shown clearly by the observation of substrate inhibition by 5-aminosalicylic acid, the correlation of calculated and experimental self-consistency factors (den vs. den calc ) and the F-test comparisons. Therefore, the theoretical model which best fits the experimental data is a Ping Pong Bi Bi mechanism with substrate inhibition by the acceptor substrate 5-aminosalicylic acid on both the free form of the enzyme and the enzyme·AcCoA complex.
Determination of the half-life of the acetylated enzyme intermediate
Determination of the half-life of the acetyl-enzyme intermediate.
Rate of hydrolysis of acetyl donor (nM·s-1) a
k3 (× 10-3·s-1) b
t1/2 (s) c
4 mM PANAT
8 mM PANAT
0.28 ± 0.01
12.2 ± 0.5
22.8 ± 0.4
2.83 ± 0.08
235 ± 8
21.2 ± 3.9
2.66 ± 0.49
270 ± 49
The rates of enzyme-catalysed hydrolysis of p-nitrophenyl acetate were 12.2 ± 0.5 nM·s-1 for 4 μM PANAT and 22.8 ± 0.4 nM·s-1 for 8 μM PANAT after correction for the non-enzymatic rate of reaction, which was 0.28 ± 0.01 nM·s-1. These results give a k3 value of 2.83 ± 0.08 × 10-3 s-1, which corresponds to a value for t1/2 of the acetyl-NAT intermediate of 235 ± 8 s.
The rate of enzyme-catalysed hydrolysis of AcCoA was 21.2 ± 3.9 nM·s-1 for 8 μM PANAT. Under the experimental conditions used, no hydrolysis of AcCoA was observed in the absence of enzyme. The calculated k3 was 2.66 ± 0.49 × 10-3 s-1, corresponding to a value for t1/2 of acetyl-PANAT of 270 ± 49 s. Thus, the half-life of the acetyl-PANAT intermediate is very similar when generated with p-nitrophenyl acetate or AcCoA as the acetyl donor.
Comparison of AcCoA and p-nitrophenyl acetate as acetyl donors
Comparison of PANAT-catalysed N-acetylation with p-nitrophenyl acetate or AcCoA as acetyl donora
Specific Activity – PNPA (nmol·min-1·mg-1) b
Specific Activity – AcCoA (nmol·min-1·mg-1) c
1040 ± 30
73300 ± 3300
1470 ± 40
44710 ± 2720
2990 ± 10
29550 ± 3110
841 ± 17
8200 ± 78
2220 ± 40
13500 ± 0
602 ± 3
2324 ± 0
567 ± 7
629 ± 40
When the acetyl donor p-nitrophenyl acetate is used, the product of the initial enzyme acetylation step is p-nitrophenol. The rate of the PANAT-catalysed acetylation of 5-aminosalicylic acid with either of the acetyl donors p-nitrophenyl acetate or AcCoA was not changed in the presence of p-nitrophenol at concentrations up to 100 μM. Therefore, it is unlikely that the slower rate of reaction with p-nitrophenyl acetate compared to AcCoA observed in the present study is due to product inhibition by the p-nitrophenol produced in the first half of the Ping Pong Bi Bi reaction. When AcCoA is used as the acetyl donor, CoA is the product of the initial enzyme-acetylation step. Andres and colleagues noted marked product inhibition by CoA , which may explain the reported results with the STNAT enzyme and NAT from pigeon livers, where the rates of acetylation of acceptor substrates were faster when p-nitrophenyl acetate was used instead of AcCoA as the acetyl donor [23, 38]. Product inhibition by CoA does not explain the results obtained in this study with the PANAT enzyme; however, it is possible that the 71-fold slower acetylation of 5-aminosalicylic acid with p-nitrophenyl acetate as the acetyl donor instead of AcCoA may be due to the substrate inhibition exhibited by 5-aminosalicylic acid. As shown in this study, 5-aminosalicylic acid binds to the free form of the enzyme and the enzyme·AcCoA complex, both of which are inhibitory pathways in the proposed reaction mechanism (Figure 2). As 5-aminosalicylic acid is structurally more similar to p-nitrophenyl acetate than to AcCoA, it is possible that the effects of the observed substrate inhibition mechanisms would be greater when p-nitrophenyl acetate is used as the acetyl donor instead of AcCoA.
A summary of all kinetic constants determined for the PANAT-catalysed acetylation of 5-aminosalicylic acid
1.02 ± 0.29 mM
0.219 ± 0.073 mM
1.93 ± 0.28 mM
1.03 ± 0.34 mM
1.75 ± 0.07 μM·s-1
434 ± 17 s-1
468 ± 150 mM-1·s-1
2259 ± 830 mM-1·s-1
The first steps of any Ping Pong Bi Bi mechanism are the binding and reaction of the first substrate with the enzyme, resulting in the formation of a stable, non-native enzyme intermediate . The isolation of a stable enzyme intermediate may be taken as positive evidence for a Ping Pong mechanism over an Ordered mechanism, in which all substrates bind to the enzyme prior to the release of the first product . Figure 3 shows the generic reaction of NAT with an acetyl donor in the absence of acceptor substrate. The acetylation of hamster NAT2 with p-nitrophenyl acetate has been shown to be rapid, by stopped-flow techniques . For hamster NAT2, the rate of hydrolysis of the acetyl-enzyme intermediate was found to be rate limiting, with a k3 of 7.85 ± 0.65 × 10-3 s-1 compared with an estimated k2 of 1301 ± 720 s-1 and kobs of 44.8 s-1 , which satisfies the assumptions that k2 >> k3 and kobs >> k3 (Figure 3). We have shown that PANAT catalyses the hydrolysis of both p-nitrophenyl acetate and AcCoA in the absence of acetyl acceptor substrate (Figure 4). Apart from two C-terminal truncation mutants of STNAT , no other bacterial NAT has been reported to exhibit this arylamine-independent activity. The lack of observed arylamine-independent acetyl-donor hydrolysis is most likely due to the significantly lower specific activity of the enzyme in the absence of an acetyl acceptor compared with when an acetyl acceptor is present , meaning that a high concentration of enzyme is required in order to measure the reaction rate of AcCoA hydrolysis in the absence of an acetyl acceptor. In the present study, the specific activity of PANAT-catalysed acetyl donor hydrolysis was 15,000-fold greater in the presence of the acceptor substrate 5-aminosalicylic acid compared with the acceptor-independent specific activity.
It has not been possible to detect an acetylated-STNAT enzyme intermediate by 1H and 13C NMR experiments , and it was concluded that, if an acetyl-STNAT intermediate were formed, its half-life would be considerably shorter than the timescale of the NMR experiments (~30 min). The half-life of acetyl-PANAT intermediate as determined in the present study (235 ± 8 s, Figure 4), is considerably shorter than the timescale of the NMR experiment. Jencks and colleagues measured the half-life of the acetylated NAT from pigeon liver as less than 1 min . More recently, Wagner, Hanna and co-workers measured the half-life of the acetylated NAT2 enzyme from Syrian hamsters at 88.3 ± 8.3 s . While the half-lives of the prokaryotic and eukaryotic enzymes are of a similar order of magnitude, the results obtained in this study suggest that the acetyl-PANAT intermediate may be more stable with respect to hydrolysis than both the acetyl-NAT intermediates from Syrian hamster and pigeon liver [32, 34].
Summary of Michaelis constants for AcCoA from different NATs.
True or Apparent Km
Acetyl Acceptor d
p-anisidine (0.2 μM)
p-anisidine (2 mM)
0.466 ± 0.077
[see Additional file 2]
1.02 ± 0.29
STNAT - 11 b
0.393 ± 0.003
STNAT - 85 c
0.764 ± 0.004
Pigeon liver NAT
p-nitroaniline, pABA, pABA-Glu
Rabbit liver NAT
The apparent kinetic parameters for AcCoA have been determined previously with recombinant PANAT enzyme and p-anisidine as the acetyl acceptor at a concentration of 200 μM . In this study, we have used a higher concentration of p-anisidine (2 mM) to determine the apparent kinetic parameters for AcCoA under conditions that closer approach pseudo-first order kinetics [see Additional file 2]. The following values were obtained from a Hanes plot [see Additional file 2]: the apparent Michaelis constant for AcCoA (Km,app) of 466 ± 77 μM, apparent limiting rate (Vmax,app) of 362 ± 54 nM·s-1, apparent turnover number (kcat,app) of 17.5 ± 2.6 s-1 and apparent specificity constant (kcat,app/Km,app) of 37.6 ± 5.4 mM-1·s-1. The previous studies with a lower concentration of p-anisidine as the acceptor substrate (200 μM) gave values for Km,app of 136 μM and Vmax,app of 153 nM·s-1 . At the relatively high acceptor substrate concentration of 2 mM, the apparent Michaelis constant was less than 50% of its true value (1.02 ± 0.29 mM), as determined with the normalised plot method in this study. Considering the very hydrophobic nature of many of the known acetyl acceptor substrates of NATs , it is not surprising that, in practice, the substrate concentrations required to reach pseudo-first order kinetics are often at or beyond the limit of solubility.
A summary of reported Km values for AcCoA with various NAT enzymes is given in Table 6. The very low apparent Km values for AcCoA with STNAT and NAT from pigeon livers may not be due solely to the experimental conditions used. For the reaction with hamster NAT2, it has been suggested that the deacetylation step with p-nitroaniline is rate limiting , which results in a decrease in the measured apparent Km for AcCoA compared with the true value. In the same study, the deacetylation step with p-aminobenzoic acid or p-aminobenzoyl-L-glutamate as the acetyl acceptor was found to be partially rate-limiting, thus the apparent Michaelis constants for AcCoA when using these substrates are also lower than the true value . Therefore, it is likely that the comparatively low apparent Km values measured for STNAT  and pigeon NAT  reflect a rate-limiting deacetylation step in the presence of the acetyl acceptor substrates isoniazid, N-OH-Glu-P-1 and p-nitroaniline (Table 6).
The Michaelis constant for AcCoA with hamster NAT2 (5.94 mM) is nearly 6 times higher than the Km for AcCoA (1.02 ± 0.29 mM) determined with PANAT in the present study . This indicates that AcCoA has a greater affinity for PANAT than for hamster NAT2. It is therefore possible that the longer half-life of the acetyl-PANAT intermediate compared to that of acetyl-NAT2 from hamsters may be due to the greater stability of the PANAT·AcCoA (or the acetyl-PANAT·CoA) complex.
We have shown that NAT from Pseudomonas aeruginosa follows a Ping Pong Bi Bi kinetic mechanism with substrate inhibition, by using a normalised plot method. This is the first reported study of the kinetic mechanism of a bacterial NAT. These studies provide a basis for the understanding of the molecular interactions of small-molecule ligands with NAT enzymes, and establish a reliable, reproducible and efficient method for the determination of kinetic parameters.
Recombinant PANAT was prepared and purified as previously described . Repeated concentration and dilution was used to exchange the buffer of protein solutions from the Tris.HCl buffers used during purification to phosphate-buffered saline (PBS, 0.137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.4). Protein solutions were concentrated by using Amicon centrifugal filter devices (10,000 MW Cut-off, Millipore). The concentrations of solutions containing purified PANAT were determined spectrophotometrically with a Hitachi U2001 spectrophotometer by using the molar extinction coefficient (ε280) of 33120 M-1.cm-1 (calculated with ProtParam [43, 44]).
Normalised plot analysis
The method of Bravo and colleagues  was used to determine the true kinetic parameters of the PANAT-catalysed acetylation of 5-aminosalicylic acid with AcCoA as the acetyl donor. A flowchart describing the approach is given as supplemental material [see Additional file 1]. The kinetic parameters are defined as follows: KmA and KmB, true Michaelis constants for substrates A (AcCoA) and B (5-aminosalicylic acid) respectively; KiA, dissociation constant of the EA complex (Ordered mechanism only); Ksi(B→E), dissociation constant of the EB complex to free enzyme (E) and free substrate (B). The subscript 'si' refers to substrate inhibition by the substrate on the enzyme form indicated in brackets. The nomenclature proposed by Cleland  has been used throughout for enzyme forms and complexes; thus, E is the free form of PANAT and F is the acetyl-NAT intermediate. Non-linear regression analyses were performed by using KyPlot v2.0 beta 13  with the least-squares method.
Determination of acetyl-enzyme half-life
Assays measuring the hydrolysis of the acetyl donors p-nitrophenyl acetate and acetyl coenzyme A (AcCoA) were performed in the absence of an acetyl acceptor substrate. The assay solutions (100 μL) contained PANAT (8, 4 or 0 μM; 26.7, 13.3 or 0 μg) and either AcCoA or p-nitrophenyl acetate (320 μM) in PBS (pH 7.4) containing 5% (v/v) dimethylsulfoxide (DMSO). The reactions were started by the addition of acetyl donor (5 μL) in DMSO or PBS. The production of p-nitrophenol was followed spectrophotometrically at 405 nm in a continuous assay with a Hitachi U2001 spectrophotometer or a Tecan Sunrise 96-well plate reader; the molar extinction coefficient of p-nitrophenol at this wavelength (ε405) is 13400 M-1.cm-1, and data were collected at 30 s intervals. The rate of formation of CoA was determined spectrophotometrically (Tecan Sunrise 96-well plate reader) by following the reaction of CoA with the colorimetric agent 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB, 5 mM) in stop buffer (25 μL, 100 mM Tris.HCl, 6.4 M guanidine.HCl, pH 7.3) in a non-continuous assay as previously described [11, 12]. All assays were performed at 25°C, and rates are relative to the non-enzymatic hydrolysis of AcCoA, expressed as the mean ± standard deviation of quadruplicate measurements.
Determination of NAT-catalysed N-acetylation activity
Assays measuring the N-acetylation activity of PANAT were performed with the acetyl donors p-nitrophenyl acetate and AcCoA, and the acetyl acceptors 5-aminosalicylic acid, 2-aminofluorene, hydralazine, p-aminobenzoic acid, p-anisidine, isoniazid and aniline. Assay solutions (100 μL) contained PANAT (3.4 – 20.7 nM, 11 – 69 ng), acetyl donor (0.05 – 6.00 mM) and acetyl acceptor (0.05 – 3.00 mM) in 20 mM Tris.HCl buffer (pH 8.0) or PBS. Reactions were started by the addition of acetyl donor and the rates of production of p-nitrophenol or CoA were determined as described above. All assays were performed at 25°C, and controls were included in which no enzyme or no substrate was added. All rates are relative to the non-enzymatic hydrolysis of acetyl donor, and are expressed as the mean ± standard deviation of triplicate or quadruplicate measurements.
This work was supported by an MRC graduate studentship (IMW) and a Wellcome Trust Programme Grant (ES, WT 067887). The authors thank Patrick E. Hanna (University of Minnesota), Carston R. Wagner (University of Minnesota), Akane Kawamura (University of Oxford), D. James Sandy (University of Oxford) and Peter Mackenzie (Flinders University, Adelaide) for helpful advice and discussion.
- Watanabe M, Sofuni T, Nohmi T: 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. J Biol Chem. 1992, 267: 8429-8436.PubMedGoogle Scholar
- Floss HG, Yu TW: Lessons from the rifamycin biosynthetic gene cluster. Curr Opin Chem Biol. 1999, 3: 592-597. 10.1016/S1367-5931(99)00014-9.View ArticlePubMedGoogle Scholar
- Bhakta S, Besra GS, Upton AM, Parish T, Sholto-Douglas-Vernon C, Gibson KJC, Knutton S, Gordon S, daSilva RP, Anderton MC, Sim E: Arylamine N-acetyltransferase is required for synthesis of mycolic acids and complex lipids in Mycobacterium bovis BCG and represents a novel drug target. J Exp Med. 2004, 199: 1191-1199. 10.1084/jem.20031956.PubMed CentralView ArticlePubMedGoogle Scholar
- Sinclair JC, Delgoda R, Noble ME, Jarmin S, Goh NK, Sim E: Purification, characterization, and crystallization of an N-hydroxyarylamine O-acetyltransferase from Salmonella typhimurium. Protein Expr Purif. 1998, 12: 371-380. 10.1006/prep.1997.0856.View ArticlePubMedGoogle Scholar
- Payton M, Auty R, Delgoda R, Everett M, Sim E: Cloning and characterization of arylamine N-acetyltransferase genes from Mycobacterium smegmatis and Mycobacterium tuberculosis: increased expression results in isoniazid resistance. J Bacteriol. 1999, 181: 1343-1347.PubMed CentralPubMedGoogle Scholar
- Holton SJ, Dairou J, Sandy J, Rodrigues-Lima F, Dupret JM, Noble M, Sim E: Structure of Mesorhizobium loti arylamine N-acetyltransferase 1. Acta Crystallograph Sect F Struct Biol Cryst Commun. 2005, 61: 14-16. 10.1107/S1744309104030659.View ArticleGoogle Scholar
- Westwood IM, Holton SJ, Rodrigues-Lima F, Dupret JM, Bhakta S, Noble ME, Sim E: Expression, purification, characterization and structure of Pseudomonas aeruginosa arylamine N-acetyltransferase. Biochem J. 2005, 385: 605-612. 10.1042/BJ20041330.PubMed CentralView ArticlePubMedGoogle Scholar
- Sandy J, Mushtaq A, Kawamura A, Sinclair J, Sim E, Noble M: The structure of arylamine N-acetyltransferase from Mycobacterium smegmatis - an enzyme which inactivates the anti-tubercular drug, isoniazid. J Mol Biol. 2002, 318: 1071-1083. 10.1016/S0022-2836(02)00141-9.View ArticlePubMedGoogle Scholar
- Sinclair JC, Sandy J, Delgoda R, Sim E, Noble ME: Structure of arylamine N-acetyltransferase reveals a catalytic triad. Nat Struct Biol. 2000, 7: 560-564. 10.1038/76783.View ArticlePubMedGoogle Scholar
- Sandy J, Mushtaq A, Holton SJ, Schartau P, Noble ME, Sim E: Investigation of the catalytic triad of arylamine N-acetyltransferases: essential residues required for acetyl transfer to arylamines. Biochem J. 2005, 390: 115-123. 10.1042/BJ20050277.PubMed CentralView ArticlePubMedGoogle Scholar
- Brooke EW, Davies SG, Mulvaney AW, Pompeo F, Sim E, Vickers RJ: An approach to identifying novel substrates of bacterial arylamine N-acetyltransferases. Bioorg Med Chem. 2003, 11: 1227-1234. 10.1016/S0968-0896(02)00642-9.View ArticlePubMedGoogle Scholar
- Riddles PW, Blakeley RL, Zerner B: Reassessment of Ellman's reagent. Methods Enzymol. 1983, 91: 49-60.View ArticlePubMedGoogle Scholar
- Brooke EW, Davies SG, Mulvaney AW, Okada M, Pompeo F, Sim E, Vickers RJ, Westwood IM: Synthesis and in vitro evaluation of novel small molecule inhibitors of bacterial arylamine N-acetyltransferases (NATs). Bioorg Med Chem Lett. 2003, 13: 2527-2530. 10.1016/S0960-894X(03)00484-0.View ArticlePubMedGoogle Scholar
- Hein DW: Molecular genetics and function of NAT1 and NAT2: role in aromatic amine metabolism and carcinogenesis. Mutat Res. 2002, 506-507: 65-77.View ArticlePubMedGoogle Scholar
- Kadlubar FF, Badawi AF: Genetic susceptibility and carcinogen-DNA adduct formation in human urinary bladder carcinogenesis. Toxicol Lett. 1995, 82-83: 627-632. 10.1016/0378-4274(95)03507-9.View ArticlePubMedGoogle Scholar
- Hanna PE: N-acetyltransferases, O-acetyltransferases, and N,O-acetyltransferases: enzymology and bioactivation. Adv Pharmacol. 1994, 27: 401-430.View ArticlePubMedGoogle Scholar
- Minchin RF: Acetylation of p-aminobenzoylglutamate, a folic acid catabolite, by recombinant human arylamine N-acetyltransferase and U937 cells. Biochem J. 1995, 307: 1-3.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang N, Liu L, Liu F, Wagner CR, Hanna PE, Walters KJ: NMR-based model reveals the structural determinants of mammalian arylamine N-acetyltransferase substrate specificity. J Mol Biol. 2006, 363: 188-200. 10.1016/j.jmb.2006.08.026.View ArticlePubMedGoogle Scholar
- Rodrigues-Lima F, Delomenie C, Goodfellow GH, Grant DM, Dupret JM: Homology modelling and structural analysis of human arylamine N-acetyltransferase NAT1: evidence for the conservation of a cysteine protease catalytic domain and an active-site loop. Biochem J. 2001, 356: 327-334. 10.1042/0264-6021:3560327.PubMed CentralView ArticlePubMedGoogle Scholar
- Rodrigues-Lima F, Dupret JM: 3D model of human arylamine N-acetyltransferase 2: structural basis of the slow acetylator phenotype of the R64Q variant and analysis of the active-site loop. Biochem Biophys Res Commun. 2002, 291: 116-123. 10.1006/bbrc.2002.6414.View ArticlePubMedGoogle Scholar
- Kawamura A, Graham J, Mushtaq A, Tsiftsoglou SA, Vath GM, Hanna PE, Wagner CR, Sim E: Eukaryotic arylamine N-acetyltransferase. Investigation of substrate specificity by high-throughput screening. Biochem Pharmacol. 2005, 69: 347-359. 10.1016/j.bcp.2004.09.014.View ArticlePubMedGoogle Scholar
- Payton M, Mushtaq A, Yu TW, Wu LJ, Sinclair J, Sim E: Eubacterial arylamine N-acetyltransferases - identification and comparison of 18 members of the protein family with conserved active site cysteine, histidine and aspartate residues. Microbiology. 2001, 147: 1137-1147.View ArticlePubMedGoogle Scholar
- Riddle B, Jencks WP: Acetyl-coenzyme A: arylamine N-acetyltransferase. Role of the acetyl-enzyme intermediate and the effects of substituents on the rate. J Biol Chem. 1971, 246: 3250-3258.PubMedGoogle Scholar
- Andres HH, Klem AJ, Szabo SM, Weber WW: New spectrophotometric and radiochemical assays for acetyl-CoA: arylamine N-acetyltransferase applicable to a variety of arylamines. Anal Biochem. 1985, 145: 367-375. 10.1016/0003-2697(85)90376-8.View ArticlePubMedGoogle Scholar
- Jenne JW, Boyer PD: Kinetic characteristics of the acetylation of isoniazid and p-aminosalicylic acid by a liver-enzyme preparation. Biochim Biophys Acta. 1962, 65: 121-127. 10.1016/0006-3002(62)90155-5.View ArticlePubMedGoogle Scholar
- Weber WW, Cohen SN: N-acetylation of drugs: isolation and properties of an N-acetyltransferase from rabbit liver. Mol Pharmacol. 1967, 3: 266-273.PubMedGoogle Scholar
- Cleland WW: The kinetics of enzyme-catalyzed reactions with two or more substrates or products. I. Nomenclature and rate equations. Biochim Biophys Acta. 1963, 67: 104-137. 10.1016/0006-3002(63)91800-6.View ArticlePubMedGoogle Scholar
- Andres HH, Klem AJ, Schopfer LM, Harrison JK, Weber WW: On the active site of liver acetyl-CoA. Arylamine N-acetyltransferase from rapid acetylator rabbits (III/J). J Biol Chem. 1988, 263: 7521-7527.PubMedGoogle Scholar
- Andres HH, Kolb HJ, Schreiber RJ, Weiss L: Characterization of the active site, substrate specificity and kinetic properties of acetyl-CoA:arylamine N-acetyltransferase from pigeon liver. Biochim Biophys Acta. 1983, 746: 193-201.View ArticlePubMedGoogle Scholar
- Andres HH, Vogel RS, Tarr GE, Johnson L, Weber WW: Purification, physicochemical, and kinetic properties of liver acetyl-CoA:arylamine N-acetyltransferase from rapid acetylator rabbits. Mol Pharmacol. 1987, 31: 446-456.PubMedGoogle Scholar
- Jenne JW: Partial purification and properties of the isoniazid transacetylase in human liver: its relationship to the acetylation of p-aminosalicylic acid. J Clin Invest. 1965, 44: 1992-2002.View ArticleGoogle Scholar
- Jencks WP, Gresser M, Valenzuela MS, Huneeus FC: Acetyl Coenzyme A : Arylamine Acetyltransferase (Measurement of the steady state concentration of the acetyl-enzyme intermediate). J Biol Chem. 1972, 247: 3756-3760.PubMedGoogle Scholar
- Hickman D, Palamanda JR, Unadkat JD, Sim E: Enzyme kinetic properties of human recombinant arylamine N-acetyltransferase 2 allotypic variants expressed in Escherichia coli. Biochem Pharmacol. 1995, 50: 697-703. 10.1016/0006-2952(95)00182-Y.View ArticlePubMedGoogle Scholar
- Wang H, Vath GM, Gleason KJ, Hanna PE, Wagner CR: Probing the mechanism of hamster arylamine N-acetyltransferase 2 acetylation by active site modification, site-directed mutagenesis, and pre-steady state and steady state kinetic studies. Biochemistry. 2004, 43: 8234-8246. 10.1021/bi0497244.View ArticlePubMedGoogle Scholar
- Wang H, Liu L, Hanna PE, Wagner CR: Catalytic mechanism of hamster arylamine N-acetyltransferase 2. Biochemistry. 2005, 44: 11295-306. 10.1021/bi047564q.View ArticlePubMedGoogle Scholar
- Bravo IG, Busto F, De Arriaga D, Ferrero MA, Rodriguez-Aparicio LB, Martinez-Blanco H, Reglero A: A normalized plot as a novel and time-saving tool in complex enzyme kinetic analysis. Biochem J. 2001, 358: 573-583.PubMed CentralView ArticlePubMedGoogle Scholar
- Mannervik B: Regression analysis, experimental error, and statistical criteria in the design and analysis of experiments for discrimination between rival kinetic models. Methods Enzymol. 1982, 87: 370-390.View ArticlePubMedGoogle Scholar
- Mushtaq A, Payton M, Sim E: The COOH terminus of arylamine N-acetyltransferase from Salmonella typhimurium controls enzymic activity. J Biol Chem. 2002, 277: 12175-12181. 10.1074/jbc.M104365200.View ArticlePubMedGoogle Scholar
- Sandy J, Holton S, Fullam E, Sim E, Noble M: Binding of the anti-tubercular drug isoniazid to the arylamine N-acetyltransferase protein from Mycobacterium smegmatis. Protein Sci. 2005, 14: 775-782. 10.1110/ps.041163505.PubMed CentralView ArticlePubMedGoogle Scholar
- Delgoda R, Lian LY, Sandy J, Sim E: NMR investigation of the catalytic mechanism of arylamine N-acetyltransferase from Salmonella typhimurium. Biochim Biophys Acta. 2003, 1620: 8-14.View ArticlePubMedGoogle Scholar
- Delgoda R: A Study of Arylamine N-acetyltransferase from Salmonella typhimurium. D Phil Thesis. 1999, Oxford, University of OxfordGoogle Scholar
- Westwood IM, Kawamura A, Fullam E, Russell AJ, Davies SG, Sim E: Structure and mechanism of arylamine N-acetyltransferases. Curr Top Med Chem. 2006, 6: 1641-1654. 10.2174/156802606778108979.View ArticlePubMedGoogle Scholar
- Expasy ProtParam tool. [http://us.expasy.org/tools/protparam.html]
- Gasteiger E, Hoogland C, Gattiker A, Duvaud A, Wilkins M, Appel RD, Bairoch A: Protein Identification and Analysis Tools on the ExPASy Server. The Proteomics Protocols Handbook. Edited by: Walker JM. 2005, Totowa, Humana Press, 571-607.View ArticleGoogle Scholar
- KyPlot v2.0 beta 13. [http://www.woundedmoon.org/win32/kyplot.html]
- Guex N, Peitsch MC: SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis. 1997, 18: 2714-2723. 10.1002/elps.1150181505.View ArticlePubMedGoogle Scholar
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