Identification and Characterization of Functional Rat Arylamine <i>N</i>-Acetyltransferase 3: Comparisons with Rat Arylamine <i>N</i>-Acetyltransferases 1 and 2

Walraven, Jason M.; Doll, Mark A.; Hein, David W.

doi:10.1124/jpet.106.108399

Cited by 36 publications

(51 citation statements)

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“…Previous recombinant protein expression experiments have thus far indicated that only AF (Kelly and Sim, 1994) and 5-AS (Estrada-Rodgers et al, 1998) show any measurable N-acetylation by Nat3. In contrast, the recently cloned rat Nat3 displays both functional N-and O-acetylation activity for a number of substrates when expressed in a bacterial system (Walraven et al, 2006). However, determination of the relative functional contribution of Nat3-mediated acetylation to the whole animal is difficult to assess without subtype-selective substrates because Nat3 activity could be masked by Nat1 or Nat2 activity.…”

Section: Discussionmentioning

confidence: 99%

“…Only a few substrates, namely, 5-aminosalicylic acid (5-AS) and 2-aminofluorene (AF), appear to be acetylated by Nat3 at low levels in such systems (Kelly and Sim, 1994;Estrada-Rodgers et al, 1998), and Nat3 mRNA has only been detected in spleen (Boukouvala et al, 2002). Recently, a third Nat showing 91% nucleotide identity to mouse Nat3 has been cloned from rat (Walraven et al, 2006). The rat Nat3, however, can Nacetylate a number of substrates, including AF, 4-aminobiphenyl (ABP), 5-AS, 3-ethylaniline, 3,5-dimethylaniline, and 4,4Ј-methylenedianiline, as well as O-acetylate N-hydroxy-ABP.…”

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confidence: 99%

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Effect of Arylamine AcetyltransferaseNat3Gene Knockout onN-Acetylation in the Mouse

Sugamori¹,

Brenneman²,

Wong³

et al. 2007

Drug Metab Dispos

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ABSTRACT:Arylamine N-acetyltransferases (NAT) catalyze the biotransformation of many important arylamine drugs and procarcinogens. NAT can either detoxify or activate procarcinogens, complicating the manner in which these enzymes may participate in enhancing or preventing toxic responses to particular agents. Mice possess three NAT isoenzymes: Nat1, Nat2, and Nat3. Whereas Nat1 and Nat2 can efficiently acetylate many arylamines, few substrates appear to be appreciably metabolized by Nat3. We generated a Nat3 knockout mouse strain and used it along with our double Nat1/2(؊/؊) knockout strain to further investigate the functional role of Nat3. Nat3(؊/؊) mice showed normal viability and reproductive capacity. Nat3 expression was very low in wild-type animals and completely undetectable in Nat3(؊/؊) mice. In contrast, greatly elevated expression of Nat3 transcript was observed in Nat1/2(؊/؊) mice. We used a transcribed marker polymorphism approach to establish that the increased expression of Nat3 in Nat1/2(؊/؊) mice is a positional artifact of insertion of the phosphoglycerate kinase-neomycin resistance cassette in place of the Nat1/Nat2 gene region and upstream of the intact Nat3 gene, rather than a biological compensatory mechanism. Despite the increase in Nat3 transcript, the N-acetylation of p-aminosalicylate, sulfamethazine, 2-aminofluorene, and 4-aminobiphenyl was undetectable either in vivo or in vitro in Nat1/2(؊/؊) animals. In parallel, no difference was observed in the in vivo clearance or in vitro metabolism of any of these substrates between wild-type and Nat3(؊/؊) mice. Thus, Nat3 is unlikely to play a significant role in the N-acetylation of arylamines either in wild-type mice or in mice lacking Nat1 and Nat2 activities.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Effect of Arylamine AcetyltransferaseNat3Gene Knockout onN-Acetylation in the Mouse

Sugamori¹,

Brenneman²,

Wong³

et al. 2007

Drug Metab Dispos

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“…There is an 82% identity at the amino acid level between mouse NAT2 and human NAT1 94 . In 2006, Walraven et al 95 identified and characterised a third rat NAT gene (NAT3).…”

Section: Interspecies Differences In Natsmentioning

confidence: 99%

Phase Ii Drug Metabolizing Enzymes

Jančová¹,

Anzenbacher²,

Anzenbacherová³

2010

Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub

477

347

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“…The expected nucleotide sequence was confirmed by automated sequencing of sense and antisense strands as described previously (Walraven et al, 2006) using human NAT2-specific sequencing primers (Table 1), before transformation and expression in JM105 strain Escherichia coli as described previously (Chung and Miller, 1988;Chung et al, 1989;Doll and Hein, 1995). Bacteria were lysed by sonication in homogenization buffer as described previously (Doll and Hein 1995), and total bacterial lysate protein concentration was determined for each expression (Bradford, 1976).…”

Section: Methodsmentioning

confidence: 99%

Computational and Experimental Analyses of Mammalian ArylamineN-Acetyltransferase Structure and Function

Walraven¹,

Trent²,

Hein³

2007

Drug Metab Dispos

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ABSTRACT:Arylamine N-acetyltransferases (NATs) play an important role in the metabolism of arylamine and hydrazine drugs and many arylamine procarcinogens. The two human N-acetyltransferases, NAT1 and NAT2, are widely distributed in human tissues and are highly polymorphic. Although many xenobiotic procarcinogens and drugs are known mammalian NAT substrates, it is unclear what physiological roles these enzymes might play, what endogenous substrates they primarily act upon, or the mechanisms underlying the functional effects of specific NAT gene coding region single-nucleotide polymorphisms. Analyses of mammalian NAT protein structures can greatly help to answer these questions. Homology modeling techniques can be used to approximate mammalian NAT structures using known bacterial NAT crystal structures as templates. In comparison to the bacterial template NATs used for homology modeling, mammalian NATs have a 17-residue insert of unknown structure and function. Homology modeling analyses yielded two different alignments (Modeler 8v1 or 3DCof-fee algorithms) that placed this insert in two likely alternative locations. Secondary structure prediction techniques and experimental analyses of a series of human NAT2 mutants with artificial deletions/replacements of the insert region distinguished one of these alternatives as the most likely insert location and provided a better understanding of its structure and function. This study demonstrates both the utility and limitations of computational structural modeling with proteins that differ as much as the mammalian and bacterial NATs.Arylamine N-acetyltransferases (NATs; EC 2.3.1.5) catalyze the N-acetylation of arylamines and hydrazines and O-acetylation of N-hydroxy-arylamines and heterocyclic amines. These reactions are important for the activation and deactivation of exocyclic aminecontaining procarcinogens, and for the metabolism of some pharmaceutical drugs (Weber and Hein, 1985). In a ping-pong bi-bi reaction mechanism, the enzyme first acetylates the active site cysteine using acetyl-coenzyme A, and then transfers the acetyl group to the substrate's exocyclic nitrogen (N-acetylation) or the oxygen of its oxidized exocyclic nitrogen (O-acetylation) (Hein, 1988). Whereas Nacetylation is typically considered a deactivation step, O-acetylation is an activation step, resulting in the formation of reactive arylnitrenium species that can react with DNA to form adducts (Hanna, 1996). Because human NAT genes are highly polymorphic, it is important to understand how different NAT genotypes alter N-acetylation phenotypes, thereby influencing cancer susceptibilities and pharmaceutical drug toxicities (Hein et al., 2000).Expression of human NAT1 and NAT2 is widely distributed throughout body tissues (Barker et al., 2006;Husain et al., 2007). Although many xenobiotic NAT substrates have been discovered, only one potential endogenous substrate, p-aminobenzoylglutamate, has been discovered (Minchin, 1995). Therefore, it is highly probable that other, yet unknown endogenous NAT s...

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Identification and Characterization of Functional Rat Arylamine N-Acetyltransferase 3: Comparisons with Rat Arylamine N-Acetyltransferases 1 and 2

Cited by 36 publications

References 37 publications

Effect of Arylamine AcetyltransferaseNat3Gene Knockout onN-Acetylation in the Mouse

Effect of Arylamine AcetyltransferaseNat3Gene Knockout onN-Acetylation in the Mouse

Phase Ii Drug Metabolizing Enzymes

Computational and Experimental Analyses of Mammalian ArylamineN-Acetyltransferase Structure and Function

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