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Arylamine N-acetyltransferase (NAT) activity in Pseudomonas aeruginosa was inhibited by ellagic acid (EA), a naturally occurring dietary plant phenol. By measuring the acetylation of 2-aminofluorene (2-AF), the NAT activity was determined. In P. aeruginosa ATCC 27853, a NAT activity of 1.37 +/- 0.25 nmol/min/10(10) CFU for intact cell and a NAT activity of 5.92 +/- 0.20 nmol/min/mg protein for cytosolic preparation were measured. EA (ranging from 1 to 0.125 mM) showed a dose-dependent inhibition of NAT activities in the analysis of both intact cell and cytosolic preparations. Enzymatic kinetics were determined and found that EA was a potent non-competitive inhibitor of NAT activity in P. aeruginosa ATCC 27853. EA inhibition of NAT activities in P. aeruginosa ATCC 27853 was time-dependent for at least 4 hrs. These data strongly indicated that EA could suppress NAT activity in P. aeruginosa.

The possible effects of naturally occurring plant phenolics, caffeic acid (CA), chlorogenic acid (CGA) and ferulic acid (FA) on arylamine N-acetyltransferase (NAT) activities on human gastrointestinal microflora, Escherichia coli, Klebsiella pneumoniae, Enterobacter aerogenes, Citrobacter koseri and Pseudomonas aeruginosa, were examined. The bacterial NAT activities were determined by HPLC measuring the acetylation of 2-aminofluorene (2-AF). Among all examined bacteria, P. aeruginosa exerted the highest NAT activity while C. koseri possessed the lowest NAT activity. CA, CGA and FA could suppress the bacterial NAT activities dose-dependently both in the intact cell and cytosolic fraction analysis. According to the analysis of kinetic parameters in E. coli and P. aeruginosa, CA, CGA and FA were shown to be potent noncompetitive inhibitors of bacterial NAT activities. For the time course experiment, 4 mM of CA and FA could inhibit bacterial NAT activities for at least 4 hour but 4 mM of CGA could only significantly suppress NAT activity in E. coli for the same reaction time. These results strongly demonstrated that CA, CGA and FA inhibited NAT activities in human gastrointestinal bacteria.

N-Acetyltransferase (NAT), responsible for bioactivation and detoxification of arylamines, has been demonstrated to be widely distributed in many organisms ranging from humans to microorganisms. Using high performance liquid chromatography (HPLC) to analyze NAT activity in bacteria, the authors found that Pseudomonas aeruginosa exhibited high NAT activity with 2-aminofluorene (2-AF) as substrate. Characteristics of this bacterial NAT were further investigated. The N-acetylation catalyzed by this enzyme is an acetyl coenzyme A (AcCoA)-dependent reaction. As the concentration of AcCoA in the reaction mixture was increased, the apparent K(m) and Vmax for 2-AF increased. The K(m) and Vmax were 0.504 +/- 0.056 mM and 31.92 +/- 3.23 nmol/min/mg protein, respectively, for the acetylation of 2-AF with 0.5 mM AcCoA. The optimum pH for the enzyme activity was estimated to be around 8.5. It was active at a temperature range from 5 degrees C to 55 degrees C, with maximum activity at 37 degrees C. The enzyme activity was inhibited by divalent metal ions including Cu++, Fe++, Zn++, Ca++, Co++, Mn++, and Mg++, suggesting that a sulfhydryl group is involved in the N-acetylation activity. The three chemical modification agents, iodoacetamide, phenylglyoxal, and diethylpyrocarbonate, all exhibited a dose-, time-, and temperature-dependent inhibition effect. Preincubation of the NAT with AcCoA provided significant protection against the inhibition of iodoacetamide and diethylpyrocarbonate, but only partial protection against the inhibition of phenylglyoxal. These results indicate that cysteine, histidine, and arginine residues are essential for this bacterial enzyme activity, and the first two are likely to reside on the AcCoA binding site, but arginine residue may be located only near the AcCoA binding site. Our data demonstrate that P. aeruginosa possesses highly active N-acetyltransferase which shares a similar catalytic mechanism as that of higher organisms. These findings are very helpful for further investigating the role of arylamine NAT in this bacterial species.

Arylamine N-acetyltransferase (NAT) activities with 2-aminofluorene and p-aminobenzoic acid were determined in the bacterium Helicobacter pylori collected from peptic ulcer patients. Cytosols or suspensions of H. pylori with or without specific concentrations of rhein co-treatment showed different percentages of 2-aminofluorene and p-aminobenzoic acid acetylation. The data indicate that there was decreased NAT activity associated with increased levels of rhein in H. pylori cytosols. Inhibition of growth studies from H. pylori demonstrated that rhein elicited dose-dependent bacteriostatic activity in H. pylori cultures: i.e. the greater the concentration of rhein, the greater the inhibition of growth to H. pylori. For the cytosol and intact bacteria examination, the apparent values of Km and Vmax were decreased after co-treatment with 40 microM rhein. This report is the first demonstration of rhein inhibition of arylamine N-acetyltransferase activity and rhein inhibition of growth in the bacterium H. pylori.

Arylamine N-acetyltransferase (NAT) activity in Pseudomonas aeruginosa was inhibited by sulindac, a drug proposed for cancer prevention. By using high performance liquid chromatography, the NAT activity for acetylation of 2-aminofluorene (2-AF) was examined. The cytosolic NAT activity in P. aeruginosa ATCC 27853 was 5.32 +/- 0.36 nmol/min/mg of protein. Sulindac displayed a dose-dependent inhibition to cytosolic NAT activity in P. aeruginosa with an IC50 value of about 0.46 mM. The NAT activity measured from intact P. aeruginosa cells was 1.39 +/- 0.12 nmol/min/10(10) CFU and this activity was inhibited by sulindac in a similar dose-dependent fashion with an IC50 value of approximately 0.42 mM. Time-course experiments showed that NAT activity measured from intact P. aeruginosa cells was inhibited by sulindac for at least 3 h. Using standard steady-state kinetic analysis, it was demonstrated that sulindac was a possible noncompetitive inhibitor to NAT activity in P. aeruginosa. The results suggest that sulindac suppressed the NAT activity in P. aeruginosa.

This study was designed to assess the effect of vitamin C on arylamine N-acetyltransferase (NAT) activity in Klebsiella pneumoniae by using HPLC to measure the acetylation of 2-aminofluorene (2-AF) with and without vitamin C. Two assay systems were performed, one with intact bacterial cell suspensions, the other with S-9 fractions (9000g supernatant). It was found that vitamin C promoted NAT activity in K. pneumoniae in a dose-dependent manner in both systems. 4 and 8 mM vitamin C were selected for further studies in S-9 fractions and intact cell systems, respectively. Through a 4-hr time course study, vitamin C promoted the N-acetylation of 2-AF in both assay systems, but, the longer the reaction time lasted, the lower the promotion rate. In the kinetic studies, vitamin C increased the value of Km from 0.42+/-0.03 mM to 2.43+/-0.87 mM in S-9 fraction assays and from 0.54+/-0.03 mM to 0.85+/-0.18 mM in intact cell assays. Vitamin C also increased the apparent Vmax values from 3.5 +/-0.08 to 39.66+/-9.81 nmol/min/mg protein in S-9 fraction assays, and from 1.28+/-0.06 to 4.88+/-0.87 nmol/min/10(10) CFU in intact cell assays, for acetylation of 2-AF. In the presence of vitamin C, the NAT activity was increased from 0.58+/-0.06 to 1.34+/-0.02 nmol/min/mg protein in S-9 fractions, and from 0.18+/-0.02 to 0.40+/-0.02 nmol/min/10(10) CFU in intact cells, for acetylation of 2-AF. From the present study, it is concluded that vitamin C does promote the N-acetylation of 2-AF in K. pneumoniae. This is a first report suggesting that oral vitamin C may be involved in modifying the mutagenicity/carcinogenicity of ingested arylamines through enhancing the NAT activity of human enteric bacteria. This interaction should be pursued in future in vivo studies.

Glycyrrhizic acid, one of the proposed chemopreventive drugs, was used to inhibit arylamine N-acetyltransferase (NAT) activity in Klebsiella pneumoniae, both in cytosol and intact bacteria. The NAT activity was measured by using high-performance liquid chromatography to assay the amounts of 2-acetyl-aminofluorene and remaining 2-aminofluorene. The NAT activity in K. pneumoniae was inhibited by glycyrrhizic acid in a dose-dependent manner. The cytosol NAT activities were 0.675 +/- 0.028 nmol min(-1) mg(-1) protein for the acetylation of 2-aminofluorene without glycyrrhizic acid and 0.367 +/- 0.008 nmol min(-1) mg(-1) protein with 8 mM glycyrrhizic acid. The NAT activities measured from intact bacteria were 0.308 +/- 0.018 nmol min(-1) 10(-10) colony forming units for the acetylation of 2-aminofluorene without glycyrrhizic acid and 0.236 +/- 0.005 nmol min(-1) 10(-10) colony forming units in the presence of 8 mM glycyrrhizic acid. The inhibition of NAT activity by glycyrrhizic acid was demonstrated to remain for at least 4 h. The apparent Km and Vmax values calculated from cytosol NAT were 1.08 +/- 0.05 mM and 9.09 +/- 0.11 nmol min(-1) mg(-1) protein, respectively, for 2-aminofluorene. In the presence of 8 mM glycyrrhizic acid, the apparent Km and Vmax values were 0.15 +/- 0.01 mM and 0.95 +/- 0.11 nmol min(-1) mg(-1) protein, respectively, for 2-aminofluorene. In intact bacteria, the apparent Km and Vmax values were 1.28 +/- 0.48 mM and 4.08 +/- 1.06 nmol min(-1) 10(-10) colony forming units, respectively, for 2-aminofluorene. However, in the presence of 8 mM glycyrrhizic acid, the apparent Km and Vmax values were 0.67 +/- 0.09 mM and 1.82 +/- 0.37 nmol min(-1) 10(-10) colony forming units, respectively, for 2-aminofluorene. Taking these results together, the NAT activity in K. pneumoniae was inhibited by glycyrrhizic acid both in cytosol and intact bacteria. This study provides the first evidence to demonstrate that glycyrrhizic acid inhibits bacterial NAT activity.

Ibuprofen, one of the nonsteroidal anti-inflammatory drugs, inhibited arylamine N-acetyltransferase activity of Klebsiella pneumoniae both in vitro and in vivo. The NAT activities of Klebsiella pneumoniae were inhibited by ibuprofen in a dose-dependent manner both in vitro and in vivo. In vitro, the NAT activity was 0.675 +/- 0.028 nmol/min/mg of protein for the acetylation of 2-aminofluorene. In the presence of 8 mM ibuprofen, the NAT activity was 0.506 +/- 0.002 nmol/min/mg of protein for the acetylation of 2-aminofluorene. In vivo, the NAT activity was 0.279 +/- 0.016 nmol/min/10(10) colony forming units (CFU) for the acetylation of 2-aminofluorene. In the presence of 8 mM ibuprofen, the NAT activity was 0.228 +/- 0.008 nmol/min/10(10) CFU for the acetylation of 2-aminofluorene. The inhibition of NAT activity by ibuprofen was shown to persist for at least 4 h. For in vitro examination, the values of apparent Km and Vmax were 1.08 +/- 0.05 mM and 9.17 +/- 0.11 nmol/min/mg of protein, respectively, for 2-aminofluorene. However, when 8 mM of ibuprofen was added to the reaction mixtures, the values of apparent Km and Vmax were 1.19 +/- 0.01 mM and 6.67 +/- 0.11 nmol/min/mg of protein, respectively, for 2-aminofluorene. For in vivo examination, the values of apparent Km and Vmax were 1.24 +/- 0.48 mM and 4.18 +/- 1.06 nmol/min/10 x 10(10) CFU, respectively, for 2-aminofluorene. However, when 8 mM of ibuprofen was added to the culture, the values of apparent Km and Vmax were 0.95 +/- 0.29 mM and 2.77 +/- 0.37 nmol/min/mg protein, respectively, for 2-aminofluorene, respectively. This report is the first finding of ibuprofen inhibition of arylamine N-acetyltransferase activity in a strain of Klebsiella pneumoniae.

N-Acetyltransferase activities with p-aminobenzoic acid and 2-aminofluorene were determined in Helicobacter pylori from gastroduodenal disease patients. The N-acetyltransferase activity was determined using an acetyl CoA recycling assay and high pressure liquid chromatography. The N-acetyltransferase activities from a number of Helicobacter pylori samples were found to be 0.91 +/- 0.12 nmole/min/mg protein for the acetylation of 2-aminofluorene and 0.75 +/- 0.22 nmole/min/mg protein for the acetylation of p-aminobenzoic acid. The apparent K(m) and V(max) values obtained were 1.10 +/- 0.08 mM and 2.34 +/- 0.14 nmol/min/mg protein for 2-aminofluorene, and 0.92 +/- 0.09 mM and 2.08 +/- 0.16 nmol/min/mg protein for p-aminobenzoic acid. The optimal pH value for the enzyme activity was 6.0 for both substrates tested. The optimal temperature for enzyme activity was 37 degrees C for both substrates. The N-acetyltransferase activity was inhibited by iodacetamide: at 0.25 mM iodacetamide, activity was reduced 50% and 1.0 mM iodacetamide inhibited activity more than 90%. Among a series of divalent cations and salts, Cu2+ and Zn2+ were demonstrated to be the most potent inhibitors. Among the protease inhibitors, only ethylenediaminetetraacetic acid significantly protected N-acetyltransferase. Iodoacetic acid, in contrast to the other agents, markedly inhibited N-acetyltransferase. This is the first demonstration of acetyl CoA:arylamine N-acetyltransferase activity in Helicobacter pylori.

Arylamine N-acetyltransferase (NAT) activity in Pseudomonas aeruginosa was inhibited by ellagic acid (EA), a naturally occurring dietary plant phenol. By measuring the acetylation of 2-aminofluorene (2-AF),the NAT activity was determined. In P. aeruginosa ATCC 27853, a NAT activity of 1.37 +/- 0.25 nmol/min/1010 CFU for intact cell and a NAT activity of 5.92 +/- 0.20 nmol/min/mg protein for cytosolic preparation were measured. EA (ranging from 1 to 0.125 mM) showed a dose-dependent inhibition of NAT activities in the analysis of bothintact cell and cytosolic preparations. Enzymatic kinetics were determined and found that EA was a potent non-competitive inhibitor of NAT activity in P. aeruginosa ATCC 27853. EA inhibition of NAT activities in P. aeruginosa ATCC 27853 was time-dependent for at least 4 hrs.These data strongly indicated that EA could suppress NAT activity inP. aeruginosa. [ABSTRACT FROM AUTHOR]