Molecular identification of NAT8 as the enzyme that acetylates cysteine S-conjugates to mercapturic acids.
Our goal was to identify the reaction catalyzed by NAT8 (N-acetyltransferase 8), a putative N-acetyltransferase homologous to the enzyme (NAT8L) that produces N-acetylaspartate in brain. The almost exclusive expression of NAT8 in kidney and liver and its predicted association with the endoplasmic reticulum suggested that it was cysteinyl-S-conjugate N-acetyltransferase, the microsomal enzyme that catalyzes the last step of mercapturic acid formation. In agreement, HEK293T extracts of cells overexpressing NAT8 catalyzed the N-acetylation of S-benzyl-L-cysteine and leukotriene E(4), two cysteine conjugates, but were inactive on other physiological amines or amino acids. Confocal microscopy indicated that NAT8 was associated with the endoplasmic reticulum. Neither of the two frequent single nucleotide polymorphisms found in NAT8, E104K nor F143S, changed the enzymatic activity or the expression of the protein by >or=2-fold, whereas a mutation (R149K) replacing an extremely conserved arginine suppressed the activity. Sequencing of genomic DNA and EST clones corresponding to the NAT8B gene, which resulted from duplication of the NAT8 gene in the primate lineage, disclosed the systematic presence of a premature stop codon at codon 16. Furthermore, truncated NAT8B and NAT8 proteins starting from the following methionine (Met-25) showed no cysteinyl-S-conjugate N-acetyltransferase activity when transfected in HEK293T cells. Taken together, these findings indicate that NAT8 is involved in mercapturic acid formation and confirm that NAT8B is an inactive gene in humans. NAT8 homologues are found in all vertebrate genomes, where they are often encoded by multiple, tandemly repeated genes as many other genes encoding xenobiotic metabolism enzymes.
Veiga-da-Cunha
Maria
M
Laboratory of Physiological Chemistry, de Duve Institute and Université Catholique de Louvain, Avenue Hippocrate 75, B-1200 Brussels, Belgium. maria.veiga@uclouvain.be
Tyteca
Donatienne
D
Stroobant
Vincent
V
Courtoy
Pierre J
PJ
Opperdoes
Fred R
FR
Van Schaftingen
Emile
E
D000105:Acetyl Coenzyme A / Q000737:chemistry; D000107:Acetylation; D000111:Acetylcysteine / Q000737:chemistry*; D000123:Acetyltransferases / Q000737:chemistry* / Q000235:genetics; D002460:Cell Line; D018388:Codon, Terminator; D003545:Cysteine / Q000737:chemistry*; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006801:Humans; D018613:Microscopy, Confocal; D009154:Mutation; D020641:Polymorphism, Single Nucleotide; D015262:Xenobiotics / Q000737:chemistry
D018388:Codon, Terminator; D015262:Xenobiotics; D000105:Acetyl Coenzyme A; D000123:Acetyltransferases; C116373:NAT8 protein, human; D003545:Cysteine; D000111:Acetylcysteine
Anal Biochem. 1976 May 7;72:248-54
942051
Dev Biol. 2001 Jun 15;234(2):483-96
11397015
Eur J Biochem. 1990 Nov 26;194(1):309-15
2174780
Methods Enzymol. 2005;400:531-42
16399368
Trends Genet. 2007 May;23(5):219-24
17382428
Toxicol Lett. 2001 Oct 15;124(1-3):21-36
11684355
Biochem J. 1975 May;147(2):283-9
241322
Traffic. 2006 May;7(5):589-603
16643281
Biochem J. 2007 Aug 1;405(3):379-95
17623009
Biol Chem. 1997 Aug;378(8):787-91
9377473
J Cell Biol. 2007 Dec 17;179(6):1193-204
18086916
Amino Acids. 2006 Feb;30(1):1-15
16463021
Science. 1985 Oct 18;230(4723):330-2
4048937
Biol Chem. 2003 Feb;384(2):213-8
12675513
Toxicol Appl Pharmacol. 2001 Dec 1;177(2):85-93
11740907
Biochem J. 2009 Dec 14;425(1):127-36
19807691
J Biol Chem. 1986 Jan 15;261(2):766-71
3941100
BMC Evol Biol. 2007 Mar 23;7:46
17381843
BMC Bioinformatics. 2006 Oct 06;7:436
17026747
BMC Med Genet. 2008 Apr 10;9:25
18402670
J Biol Chem. 1990 Dec 15;265(35):21771-8
2174886
Biochem Pharmacol. 1998 Sep 15;56(6):763-71
9751082
Genome Biol. 2004;5(8):401
15287973
J Cell Biol. 1991 May;113(4):779-91
1827445
Bioinformatics. 2001 Aug;17(8):754-5
11524383
Arch Biochem Biophys. 2008 Jun 1;474(1):72-81
18342615
PLoS Genet. 2007 May 11;3(5):e67
17500592
J Hum Genet. 1998;43(4):255-8
9852678
Ann N Y Acad Sci. 1988;524:35-42
2837969
Mol Biol Cell. 1995 Oct;6(10):1315-32
8573789
Proc Natl Acad Sci U S A. 2004 Nov 30;101(48):16849-54
15548604
Eur J Biochem. 1984 Jul 16;142(2):281-6
6745276
FASEB J. 1997 Jan;11(1):3-14
9034160
J Biol Chem. 1988 Feb 25;263(6):2773-8
2830262
Mol Cell Biol. 2006 May;26(9):3353-64
16611980
Biochem J. 1996 Jul 1;317 ( Pt 1):213-8
8694766
Toxicology. 1993 Mar 30;79(1):67-79
8475500
Nucleic Acids Res. 1997 Dec 15;25(24):4876-82
9396791
J Biol Chem. 2009 Jan 23;284(4):2482-92
19011241