Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK1
Department of Chemistry, Box 351700, University of Washington, Seattle, WA 98195-1700, USA2
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK3
The Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK4
Author for correspondence: Edith Sim. Tel: +44 1865 271596. Fax: +44 1865 271853. e-mail: esim{at}molbiol.ox.ac.uk
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ABSTRACT |
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Keywords: gene cluster, NAT, prokaryotic, endogenous function
Abbreviations: NAT, arylamine N-acetyltransferase encoded by nat in prokaryotes and NAT in eukaryotes; INH, isoniazid; p-ABA, p-aminobenzoic acid; RAS, rifamycin amide synthase
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INTRODUCTION |
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The Ames tester strains of Salmonella typhimurium (TA98 and XG1024) are used to predict the mutagenic properties of aromatic amines and nitroarenes. Sensitivity to these compounds is through N-hydroxylamine O-acetyltransferase activity and was the first identification of bacterial NAT (McCoy et al., 1983 ; Watanabe et al., 1992
). Since then nat, and associated acetylation activity, has been found in the Mycobacterium smegmatis and Mycobacterium tuberculosis (Payton et al., 1999
). The pathogenic mycobacterium M. tuberculosis is the main cause of tuberculosis in man (http://www.who.int/gtb/index.htm) and the choice of therapy is often INH. An increased production of NAT in mycobacteria results in an increased tolerance to INH (Payton et al., 1999
). The study of NAT has once again become associated with the anti-tubercular agent INH. NAT activity has also been reported in Escherichia coli (Chang & Chung, 1998
).
NAT is highly conserved in the eukaryotic kingdom and this study was undertaken to determine how widespread NAT is in the prokaryotic kingdom and to what extent conserved residues are maintained. Genes transcribed in bacteria often appear as related operons. Characterization of these bacterial operons may lead to a better understanding of the endogenous function of NATs in prokaryotes.
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METHODS |
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Isolation and cloning of prokaryotic nat.
The N-terminal 175 aa of Sal. typhimurium NAT was used to identify NAT-like sequences from forward and reverse translations of bacterial genomes with electronic database searches (http://www.tigr.org/tdb/mdb/mdb.html) using BLAST and FASTA3 programs (http://www.ebi.ac.uk/fasta3/). Sequences representative of the mycobacteria M. smegmatis and M. tuberculosis were determined as described previously (Payton et al., 1999 ). DNA representing the ORF of nat from all described prokaryotes was amplified by PCR using pfu DNA polymerase (Stratagene) supplemented with 6% DMSO for the GC-rich mycobacterial genomes. Products were ligated into PCR-Script (Stratagene) for sequence analysis and were subcloned to yield cleavable histidine tags in the vectors pET28b (2·3 kDa tag) or pRSET (3·1 kDa tag) (Invitrogen) for protein expression. The mycobacterial nat sequences were also subcloned into the mycobacterial expression vector pACE-1 (Parish et al., 1997
).
The endogenous copies of M. smegmatis and B. subtilis nat were disrupted using either kanamycin or ß-galactosidase cassettes inserted 5' to the first region of conservation (Fig. 1 using technology described by Hinds et al. (1999)
and Sambrook et al. (1989)
.
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Recombinant protein encoded by M. smegmatis and M. tuberculosis nat was obtained using the mycobacterial expression vector pACE-1 and the host M. smegmatis as described previously (Payton et al., 1999 ).
Western blot analysis.
Protein samples were separated by SDS-PAGE and transferred to a nylon support (Roche) for Western blot analysis. Antisera were raised in rabbits and generated against purified recombinant NAT proteins isolated in SDS-PAGE gel slices representative of Sal. typhimurium NAT. Unless otherwise stated, antisera were used at a dilution of 1:100000. Bound rabbit immunoglobulin was detected by chemiluminescence using mouse anti-rabbit IgG mAbs (used at a dilution of 1:12000) conjugated to horseradish peroxidase (Sigma). Equal standardized amounts of soluble protein were added in all cases. The protein loaded onto gels was prepared from the same number of cells as determined by optical density at 600 nm prior to lysis to ensure equal loading.
Enzymic assays.
All enzymic reactions were carried out under conditions where the initial rate was linear. Michaelis constants and specific activities for N-acetyltransferase were measured from soluble cell fractions and pure recombinant protein preparations as described previously (Sinclair et al., 1998 ). Acetyl-CoA (440 µM) with up to five times the Km concentration of arylamine or hydrazine substrate were used. Loss of substrate was indicative of enzyme activity and was determined either colorimetrically (Sinclair et al., 1998
) or, in the case with INH, by fluorometric detection following appropriate dilutions of the reaction mixtures (Ellard & Gammon, 1976
).
Computer analysis.
Database searches were performed with the programs FASTA and BLAST using a cut-off value of greater than 1x10-4 (http://www.ebi.ac.uk/fasta3/; http://www.hgsc.bcm.tmc.edu/SearchLauncher/). ORF prediction and manipulation was executed using DNA Strider (DNA Strider 1.2.1, CEA France) and NIX (http://www.hgmp.mrc.ac.uk/) in combination with homology searches (http://www.hgsc.bcm.tmc.edu/SearchLauncher/). Alignment of predicted amino acid sequences and homology shading was performed using the European Bioinformatics Institute server (http://www.ebi.ac.uk/fasta3/) and MacBoxshade (http://www.netaxs.com/~jayfar/mops.html).
RNA isolation and Northern analysis.
Total RNA was prepared from M. smegmatis, E. coli and B. subtilis using Catrimox-14 (Iowa Biotechnology) as described previously (Payton & Pinter, 1999 ). RNA (20 µg) was size-fractionated by electrophoresis on a formaldehyde denaturing 0·9% agarose gel (Sambrook et al., 1989
) followed by pre-treatment with 0·05 M NaOH to hydrolyse RNA and ensure effective transfer of large transcripts to the nylon filter (Roche). RNA markers (Promega) were treated in an identical manner to the RNA samples. Probes representing the corresponding bacterial nat ORFs (see Table 1
for primers used) were generated by PCR amplification using a molar ratio of 1:1 dTTP/DIG-labelled dUTP (Roche) to ensure effective detection of rare transcripts. Nylon filters were probed for 16 h at 50 °C in DIG Easy Hyb buffer (Roche), then washed twice for 5 min in 2xSSC/0.1% SDS (room temperature) and twice for 15 min in 0·1xSSC/0·1% SDS (50 °C). Signal detection was according to the manufacturers recommendations and filters were exposed to autoradiographic film (Sigma) for between 20 min and 2 h.
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RESULTS |
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Homologous regions within prokaryotic NAT
Sequence comparison of eukaryotic and prokaryotic NAT demonstrates a higher degree of conservation towards the N termini of these proteins compared to the C-terminal 100 aa (Fig. 1). Of the previously described key residues, Arg9, Arg64 and Cys68 (Delomenie et al., 1997
; Watanabe et al., 1992
) in the human NAT isoenzymes, Cys68 is the only amino acid to be completely retained in the prokaryotic homologues. Structural predictions and crystallographic analysis of Sal. typhimurium NAT previously revealed the existence of three domains (Fig. 1
) and an active site catalytic triad (asterisks; Fig. 1
) consisting of Cys69-His107-Asp122 (Hubbard et al., 1996
; Sinclair et al., 2000
). A comparison of the amino acid sequences representative of NATs from the Proteobacteria and Firmicutes (Table 2
) reveals high conservation in domains 1 and 2 (3852%), with a marked decrease in domain 3 (<2042%).
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Prokaryotic NAT enzymic activities
nat-like sequences were cloned from bacteria representative of the Proteobacteria (E. coli and Sal. typhimurium) and the Firmicutes [Act. mediterranei (Pseudocardineae), M. smegmatis and M. tuberculosis (Corynebacterineae) and B. subtilis (Bacillus/Clostridium group)] for recombinant protein production. Prokaryotic recombinant NAT isoenzymes were purified and the enzymic activities were determined using the well characterized eukaryotic NAT substrates p-aminobenzoic acid (p-ABA), which is a specific substrate for human NAT1, and INH, which is a specific substrate for human NAT2 (Fig. 4a; Andres et al., 1983
; Hickman et al., 1995
). With the substrates tested, the bacterial NATs are more similar to the human NAT2. The Km for p-ABA with all of the recombinant, purified bacterial NAT proteins from Sal. typhimurium, M. smegmatis, B. subtilis, E. coli and M. tuberculosis was greater than 5 mM, whereas activities could readily be detected with INH with each of these enzymes. The Km for the enzyme from B. subtilis was 39 µM and so is very similar to the previously reported value for the mycobacterial NAT enzymes (Payton et al., 1999
). The E. coli enzyme had a Km of 1·2 mM for INH.
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Detection of endogenous and recombinant prokaryotic NAT
Endogenous bacterial NAT enzymic activities were found in the soluble lysates of E. coli, Sal. typhimurium, B. subtilis and M. smegmatis using INH as substrate, albeit at a low level (Table 3). No detectable acetylation of INH was observed using soluble lysates of the NAT knockout mutants of M. smegmatis and B. subtilis (Table 3
). The level of endogenous activity in E. coli is much less than that measured by Chang & Chung (1998)
and no activity was detected with p-ABA. The lack of endogenous activity with p-ABA matched the observations obtained with the pure, recombinant enzymes (see above).
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nat operon analysis
Transcript sizes of approximately 7, 9 and 4 kb were associated with RNA isolated from M. smegmatis, E. coli and B. subtilis, respectively (lanes ac, Fig. 6) and corresponded well to predicted transcript sizes from whole genome analysis (Fig. 7
) Transcript sizes for the Amy. mediterranei and Str. achromogenes nat-like gene clusters have been reported (Floss & Yu, 1999
; Sohng et al., 1997
).
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DISCUSSION |
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Alignment of the primary amino acid sequence reveals three highly conserved regions (designated iiii) found in all NATs (Fig. 1). In domains 1 and 2 the three highly conserved regions (iiii) line the putative substrate pocket (Fig. 3
). The third C-terminal domain in Sal. typhimurium NAT forms a distinct
/ß lid to the molecule to create an active site cleft (Fig. 3
). It was previously shown using human NAT1 that the first two domains alone were necessary to generate an acetylated intermediate and that the third domain was essential in the transfer of the acetyl group to the acceptor amine (Sinclair & Sim, 1997
). Although these studies were performed with human NAT, they provide further evidence that the C-terminal domain is the key to arylamine substrate specificity.
Eukaryotic organisms have up to three isoenzymes of NAT encoded by distinct genes (Hein et al., 2000 ) and these multiple genes have probably arisen as a result of gene duplication. If there is only one isoform in mammals, it is the equivalent to human NAT1 which has been shown previously to acetylate p-ABA and p-aminobenzoylglutamate (p-ABGlu) (Cribb et al., 1991
; Minchin, 1995
; Ward et al., 1995
). The widespread distribution of this isoenzyme amongst eukaryotes may be related to its putative endogenous role in the N-acetylation of p-ABGlu (Minchin, 1995
; Ward et al., 1995
), a folate catabolite. Prokaryotic organisms use p-ABA to synthesize folate de novo and therefore the N-acetylation of p-ABA would be detrimental to the survival of a bacterium.
Acetyl-CoA is an important co-factor for numerous enzymes and binds to a variety of motifs (Engel & Wierenga, 1996 ). A 4 aa motif commencing with a Gly has been associated with phosphate binding to acetyl-CoA (Kinoshita et al., 1999
). There is an identifiable motif in Sal. typhimurium NAT in which Gly126 is situated adjacent to the active site Cys69 (Sinclair et al., 2000
). This Gly126 is highly conserved in all prokaryotic NAT enzymes identified in this report with the exception of the NAT-like enzymes of Amy. mediterranei (G126P), Act. pretiosum (G126A) and Str. achromogenes (G126A). If Gly126 is required for acetyl-CoA binding, the absence of this residue would suggest a loss in acetyl-CoA binding and thus explain the lack of acetylation activity found with the NAT-like enzyme of Amy. mediterranei (Table 3
). The highly conserved regions (i)(iii) (Fig. 1
) are found to be closely associated with the active site triad within the crystal structure of Sal. typhimurium NAT (Fig. 3
). In the NAT-like sequences identified in the Pseudonocardineae and Streptomycineae (Fig. 2
), an addition of up to 8 aa is seen in combination with partial disruption of region (i). This may be a key factor in the recognition of much larger substrates (Fig. 4b
) known for the NAT-like enzymes of Amy. mediterranei, Act. pretiosum and Str. achromogenes.
Eukaryotic NAT is known to activate potential carcinogens and polymorphic NAT in eukaryotes often results in the generation of variants in which acetylation rates are altered (Blum et al., 1992 ). These NAT isoenzymes have been shown to metabolize xenobiotics at different rates and have been linked to disease susceptibility (e.g. Risch et al., 1995
). Polymorphic NAT isoenzymes in the Enterobacteriaceae are of clinical interest as different strains within these genera may contribute to the activation of potential carcinogens in the human gut. Further kinetic studies will determine the effect of the mutations identified in this report on enzymic activity.
The NAT-like proteins isolated from Amy. mediterranei (Fig. 7), Act. pretiosum and Str. achromogenes (Sohng et al., 1997
) have been shown to be transcribed as part of an antibiotic synthesis gene cluster (Floss & Yu, 1999
). It has been deduced that these particular NATs may be key to the cyclization of the ansamycin precursors to yield proansamycin X (Amy. mediterranei; Yu et al., 1999
) and rubranasol (Str. achromogenes; Sohng et al., 1997
) (Fig. 4b
). This cyclization is via amide formation and the enzyme responsible for the production of proansamycin X has been accordingly termed rifamycin amide synthase (RAS; Yu et al., 1999
). RAS forms part of an immense 90 kb cluster (Fig. 7
) which encodes the proteins responsible for the complete biosynthesis of the antibiotic rifamycin B in Amy. mediterranei (Floss & Yu, 1999
; Schupp et al., 1998
; Tang et al., 1998
).
Bacterial genomes lack much of the non-coding sequence associated with the eukaryotic genome. Often eubacterial transcripts consist of related genes in an operon, as found with the rifamycin cluster of Amy. mediterranei. Whole-genome analysis of the pathogenic mycobacteria within the M. tuberculosis and M. avium complexes are revealing unexpected cellular processes such as an ability to survive anaerobically (Cole et al., 1998 ). We have found transcription of nat in M. tuberculosis by RT-PCR (not shown). It is therefore likely, as with the other predicted gene clusters in this report, that the slow-growing mycobacteria also encode a nat operon (Fig. 7
). The putative nat operon in pathogenic mycobacteria (Fig. 7
) encodes enzymes capable of metabolizing aromatic and biphenyl-based compounds. As well as generating an additional source of carbon and nitrogen, this cluster contributes to the ability of the mycobacterium to survive in a highly toxic environment. Such a finding is supported by the genome of M. tuberculosis encoding over 20 separate cytochrome P450 enzymes (Cole et al., 1998
), more than any other bacterium studied to date.
Unlike the bacterial and mycobacterial cytochrome P450 enzymes (Fulco, 1991 ; Poupin et al., 1999
), NAT production in Sal. typhimurium would appear not to be regulated by the presence of substrate, although higher concentrations of substrate may be required (Fig. 5b
, c
). It has been reported that xenobiotic enzymes are often involved in co-metabolism and therefore produced in response to a mixture of substrates or a compound involved earlier in the degradation pathway (French et al., 1999
). This may also be the case with the nat operon, implying regulation by aromatic and/or biphenyl-based compounds other than NAT ligands.
To date, approximately 33 complete prokaryotic genomes (including 5 archaeal sequences) have been determined (Fig. 2) and in excess of 90 are ongoing (http://www-fp.mcs.anl.gov/~gaasterland/genomes.html; http://www.tigr.org/tdb/mdb/mdb.html). However, only a fraction of the genomes sequenced appear to possess nat. Alignment of the primary amino acid sequences of both NAT and RAS enzymes reveals a high homology, particularly in the N-terminal domains 1 and 2 (Table 2
) and is indicative of divergent evolution from a common ancestor. The microbes shown in this report which possess nat inhabit environments such as soil and faecal material, in which there are high concentrations of aromatic compounds due to the decomposition of organic material. It is therefore possible that selection pressures on these microbes have resulted in the acquisition of genes encoding xenobiotic-metabolizing enzymes. This is supported by the gene content of the nat operons of the M. tuberculosis and M. avium complexes described in this report.
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ACKNOWLEDGEMENTS |
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Received 26 September 2000;
revised 18 December 2000;
accepted 24 January 2001.