N-hydroxyarylamine O-acetyltransferase of Salmonella typhimurium: proposal for a common catalytic mechanism of arylamine acetyltransferase enzymes.

Acetyl-CoA:N-hydroxyarylamine O-acetyltransferase is an enzyme involved in the metabolic activation of N-hydroxyarylamines derived from mutagenic and carcinogenic aromatic amines and nitroarenes. The O-acetyltransferase gene of Salmonella typhimurium has been cloned, and new Ames tester substrains highly sensitive to mutagenic aromatic amines and nitroarenes have been established in our laboratory. The nucleotide sequence of the O-acetyltransferase gene was determined. There was an open reading frame of 843 nucleotides coding for a protein with a calculated molecular weight of 32,177, which was close to the molecular weight of the O-acetyltransferase protein determined by using the maxicell technique. Only the residue of Cys69 in O-acetyltransferase of S. typhimurium and its corresponding residue (Cys68) in N-acetyltransferase of higher organisms were conserved in all acetyltransferase enzymes sequenced so far. The amino acid sequence Arg-Gly-Gly-X-Cys, including the Cys69, was highly conserved. A mutant O-acetyltransferase of S. typhimurium, which contained Ala69 instead of Cys69, no longer showed the activities of O- and N-acetyltransferase. These results suggest that the Cys69 of S. typhimurium and the corresponding cysteine residues of the higher organisms are essential for the enzyme activities as an acetyl-CoA binding site. We propose a new catalytic model of acetyltransferase for S. typhimurium and the higher organisms.

In this article, we describe the cloning of the S. typhimurium OAT gene (41). We established new Ames tester substrains highly sensitive to mutagenic aromatic amines and nitroarenes using the doned gene (42). Substrate specificity and inhibition analysis suggested that the S. typhimurium OAT is a counterpart of NAT of higher organisms. Sequence similarity of both enzymes at the amino acid level suggested that the Cys69 of the S. typhimurium OAT and the corresponding cysteine residues of the NAT of higher organisms are essential for the enzyme activities as an acetyl-CoA binding site (43). We also proposed a common catalytic mechanism of acetyltransferase for S. typhimurium and higher organisms.
Cloning of the S. typhimurium OAT Gene First, we constructed a gene library of S. typhimurium TA1538 into vector plasmid pBR322 and then selected rearranged plas-Environmental Health Perspectives mids that confer resistance to the killing effect of 2-nitrofluorene on the OAT deficient strain TA1538/1,8-DNP. One of these plasmids, pYG122 (Figure IA), had a molecular size of 11.65 kb, composed of the insert DNA (7.3 kb) and the vector plasmid pBR322. The pYG122-transformed strain YG 1007 (=TA1538/1,8-DNP(pYG122)) showed more than 10 times higher mutagenic sensitivity to 1,8dinitropyrene, 2-aminofluorene, and Glu-P-1 than did the conventional strain 0 0.2 0.8 EH V V 11 / TA1538 with vector plasmid pBR322. YG1007 had about 50 times higher Nhydroxy-Glu-P-1 OAT activity and isoniazid NAT activity than TA1538(pBR322). Thus, we concluded that pYG 122 had the OAT gene. In order to investigate the region necessary for a functional OAT, we constructed deletion derivatives of pYG 122 and found that a 1.35-kb fragment spanning from the EcoRV site (6.3 kb) to the BamHI site (7.65 kb) is necessary for OAT activity. 5 However, levels of the activity on the transformants depended on organization of the 1.35-kb insert and vectors ( Figure 1B). For example, the strain having pYG219 showed more than 10 times higher enzyme activity than did the strain having pYG218. The plasmids pYG218 and pYG219 have the same 1.35-kb DNA fragment in opposite directions. From this result, we suggested that the direction of transcription and translation of the OAT gene is from the EcoRV site to the BamHI site and the promoter for the OAT gene resides outside of the 1.35-kb fragment. The plasmids were introduced into S.typhimurium TA1538/1,8-DNP (pYG122, pYG213, pYG218, or pYG219) or E. coliXLI-Blue (pYG221) and Nhydroxy-Glu-P-1 OAT activity of cytosol fraction of the transformants was measured. Numbers represent the activity of OAT (nmole/min/mg protein). is T100 containing pBR322-ApS fYG1029 is TA100 containing pYG219. The plasmid pBR322-ApS is the same as pBR322 but its bla gene is inactivated by deletion between the first Dral site and the third Dral site of pBR322.
Nucleotide Sequence ofthe S. typhimurium OAT Gene We sequenced the cloned DNA from the PstI site (5.55 kb) to the BamHI site (7.65 kb), the region that is suggested to carry the OAT gene ( Figure 1). The result is shown in Figure Figure 2 and creates a new termination site at codon 204. From these results, we concluded that the open reading frame shown in Figure 2 is the coding region of the OAT.

The S. taphimurium OAT is a Counterpart ofNAT ofHigher
Organisms Because the OAT is present in cytosol, we have prepared the cytosol fraction of crude lysate of the cells harboring pYG221 and subjected it to enzyme assays and inhibition experiments. Table 2 shows the properties of the OAT of S. typhimurium and NATs of higher organisms for comparison. Besides N-hydroxy-Glu-P-1 OAT activity, the S. typhimurium OAT showed isoniazid and 2-aminofluorene NAT activities. On the other hand, many NATs of higher organisms also show NAT and OAT activities. The molecular weight of the S. typhimurium OAT was almost the same as that of the NATs. The sulfhydryl-blocking agents, which inhibit NAT of higher organisms (59), strongly inhibited the isoniazid NAT activity of the S. typhimurium OAT. Paraoxon, an inhibitor of N, Oacetyltransferase and deacetylase (60), did not inhibit the activity of the S. typhimurium OAT. From these observations, we suggested that the S. typhimurium OAT, the gene of which we cloned and sequenced, is a counterpart of NAT of higher organisms.   amino acids, showed 25 to 33% similarity to those of the NATs of higher organisms at the amino acid level (Figure 3). The remaining C-terminal region of the OAT had few similarities with the corresponding region of the NATs, although some similarities were observed at the nucleotide level.  LGRVILSHPASLPPRTHRLLLi7pVEDEQ% HIi4 kTAFGISLERKFVPKHGELVFTI Figure 3. Similarities in amino acid sequences between the S. typhimurium OAT and the NATs of human, rabbit, hamster, mouse, and chicken. Abbreviations used and reference papers of the sequences are as follows: S. typhimurium (S. T), this study (43); human liver monomorphic (Human-M),(23) human liver polymorphic (Human-P), (23,26) rabbit monomorphic (Rabbit-M) (30) rabbit liver polymorphic (Rabbit-P) (28,29,31) hamster liver monomorphic (Hamster-M", (32) mouse monomorphic and polymorphic (Mouse-M and P) (33) chicken liver (Chicken-L) (34) chicken pineal gland (Chicken-PG1 and PG2) (36). The deduced amino acid sequences of human liver monomorphic and rabbit monomorphic NATs are also reported by Blum et al. (25) and Sasaki et al. (28), respectively, but their sequences are different from those shown in this figure at three or one amino acids, respectively. It is reported that slow acetylator-human also has human liver polymorphic gene but there are one or two amino acid differences from the gene in rapid acetylator (23,24,27). The (19) and rabbit liver NAT (20) suggest that a cysteine residue reacts with acetyl-CoA and an activated acetyl-cysteinyl intermediate is formed. Thus, we suggest that a conserved cysteine residue among their :tionally enzymes plays an important role in the catsms and alytic mechanism as an acetyl-CoA-binding .e amino site. The similarity at the amino acid level .st that a between the S. typhimurium OAT and the ght exist NATs of higher organisms was much less cysteine than that of the NATs among the higher tivities. organisms. Hence, it was easy to focus on on liver the highly conserved regions. Among the NATs of higher organisms, three cysteine residues are conserved completely.

V K H G F T E A E L A A V M A A F D T H P E A C K
However, only one cysteine residue (Cys69 for the OAT, Cys6 for the NATs) was conserved between the S. typhimurium tGe i iYOAT and the NATs of higher organisms ( Figure 3). The amino acid sequence of R-G-G-X-C including Cys69 of the OAT or Cys of the NATs was highly conserved. The results of site-directed mutagenesis experiments indicated that a mutant 2AEI PH OAT of S. typhimurium, which contained IS Ala69 instead of Cys69, did not show any [SG V1 NAT and OAT activities (data not shown).
:SGK QVP Thus, we suggest that the Cys residue of rSG VP the OAT of S. typhimurium is essential for rSGK1 2Ip the enzyme activities as an acetyl-CoA-[SGKt 2?P binding site. It was plausible that the Cys68 residue of the NATs of higher organisms is also essential for the enzyme activities ;GKLTLTNFH because it was the only conserved residue ?SSVFITSKSF among all enzymes. Recently, Dupret and ?SSPFLKSI Grant (61) reported that among three cys-?SSVPLDKSI teine residues that were highly conserved ?VSVFVNTSF 6 ?ASVFVSTSP among NATs of higher organisms, Cys68 ?ASVFTSKSP ?DTILQKKSI was essential for the enzyme activities of ?DSLEvMCSI human liver polymorphic NAT. This ?DSLFTYSI observation supports our hypothesis that there is a common catalytic mechanism among acetyltransferase enzymes.
'TI Proposed Mechanism of Enzymatic Reactions of Acetyltransferase Enzymes Riddle and Jencks (62) showed that a general base is involved in the catalysis of pigeon liver NAT. Andres et al. (19) investigated the kinetics of NAT from pigeon liver and proposed that a basic residue is involved in a general base catalysis by attracting the proton of the cysteine residue of an acetyl-CoA-binding site. Recently, Cheon and Hanna (63) reported that an arginine residue is essential for the activity of hamster liver monomorphic and polymorphic NATs. One arginine residue, Arg 5 in the S. typhimurium OAT and Arg in the NATs of higher organisms, was highly Environmental Health Perspectives  typhimurium OAT as shown in Figure 4A.
The proton of cysteinyl group of Cys69 residue is attracted by Arg65 (or Arg ) residue, which is deprotonated even in neutral pH conditions, because the two adjacent guanidino groups of Arg64 and Arg65 provide mutual electrostatic destabilization (64). The activated Cys69 residue accepts an acetyl group from acetyl-CoA, resulting in an acetyl-cysteinyl-enzyme intermediate. Finally, this acetyl moiety is transferred to the oxygen atom of an Nhydroxyarylamine. In the transfer reaction of the acetyl group to the hydroxyamino group, the deprotonated Arg (or Arg ) again serves as a general base. We propose that the principle of this reaction mechanism is applicable to any acyltransfer reactions in both the S. typhimurium OAT and the NATs of higher organisms. The S. typhimurium OAT acts as an NAT when the acetyl moiety of the acetyl-cysteinyl intermediate is transferred to the nitrogen atom of an aromatic amine instead of the oxygen atom of an N-hydroxyarylamine. It is also proposed, with regard to mammalian NAT and OAT, that N-and 0acetyl transfer involves a common acet%ylated enzyme intermediate (12,21).
Cys 8 residues of the NATs of human, rabbit, hamster, mouse, and chicken could bind with acetyl-CoA, as for Cys69 of the S.
typhimurium OAT, whereas one of the basic amino acids, (e.g., Arg64) may be an activator ( Figure 4B). The acetyl moiety of the acetyl-Cys68 intermediate would be transferred to the nitrogen atom (N-acetylation) or the oxygen atom (O-acetylation) of an arylamine or N-hydroxyarylamine, respectively. An arylhydroxamic acid also may act as the acyl-donor instead of acetyl-CoA in the model, and the resulting activated acyl moiety would be transferred to the nitrogen atom of an arylamine (N,Nacyltransfer), or the oxygen atom of an Nhydroxyarylamine (inter and/or intramolecular N,O-acyltransfer). The S. typhimurium OAT has low but measurable N, 0-acetyltransferase activity (Igarashi et al., unpublished result). One possible reason why the S. typhimurium OAT has a low N, O-acyltransferase activity is that an arylhydroxamic acid could not easily fit in the active site where the Cys69 probably resides. However, we must point out that certain N,O-acyltransferases have different properties from the OAT and NAT: some of the enzymes are located in microsomes and are sensitive to paraoxon (60). The catalytic mechanism of these enzymes is probably different from that proposed in Figure 4A.

Future Perspective
We proposed a catalytic model for the S. typhimurium OAT and suggested that this model is applicable for NAT of higher organisms. To examine this model, biochemical and structural analyses of the purified enzyme are important. Hence we have been purifying the S. typhimurium OAT from Escherichia coli cells harboring plasmid pYG221, the cells which overproduced the OAT ( Figure 1B). Analyses of enzymatic properties of the OAT, such as demonstration of the acetyl-Cys69-enzyme intermediate by using purified enzyme, are currently being undertaken in our laboratory. For X-ray diffraction analysis, we are trying to crystallize the purified OAT.
Knowledge about acetyltransferase at the molecular level is rapidly growing. The OAT and NAT are involved in the metabolism of drugs and toxic chemicals. We hope that the new knowledge will also lead to the design of new pharmaceuticals and clinical kits, with possible human health applications.

Conclusions
We have cloned the gene of S. typhimurium OAT and established new strains highly sensitive to mutagenic aromatic amines and nitroarenes. It is suggested that the Cys69 of the S. typhimurium OAT and the corresponding cysteine residues of NAT of higher organisms are essential for the enzyme activities as an acetyl-CoA binding site. We also propose a new catalytic mechanism of acetyltransferase for S. typhimurium and higher organisms.