Characterization of naturally occurring and recombinant human N-acetyltransferase variants encoded by NAT1.
The genotype at the NAT1* locus of an interethnic population of 38 unrelated subjects was determined by direct sequencing of 1.6-kb fragments amplified by PCR. The coding exon alone and together with the 3' noncoding exon of the wild-type (NAT1*4) and the three mutant alleles (NAT1*10, *11, and *16) detected was expressed in Escherichia coli and COS-1 cells, respectively, and the cytosolic fraction of mononuclear leukocytes from NAT1*4/*4 and NAT1*10/*10 homozygotes was also isolated. Recombinant and leukocyte cytosolic preparations were thoroughly characterized by N-acetylation activity with several NAT1-specific and -selective substrates, as well as by steady-state kinetics with varying amounts of the substrate (fixed acetyl CoA) and acetyl CoA (fixed substrate), thermodynamics, stability, and protein immunoreactivity with a polyclonal human anti-NAT1. The polyadenylation signal mutation in the 3' noncoding sequence of NAT1*10 affected none of the aforementioned parameters evaluated both with recombinant NAT1*10 and with the naturally occurring allele. Function was also unaffected by the coding and 3' noncoding exon mutations in NAT1*11. In contrast, the three extra adenosines located immediately after the sixth position of the polyadenylation signal in the 3' untranslated region of NAT1*16 ostensibly caused disruption of the predicted secondary structure of the pre-mRNA for NAT1 16, culminating in parallel 2-fold decreases in the amount and catalytic activity of NAT1 16 in COS-1 cell cytosol. This novel finding in N-acetylation pharmacogenetics clearly demonstrates a direct link between reduced catalytic activity and structural alteration in the 3' untranslated region of an NAT variant (NAT1*16) brought about by mutation.
de León
J H
JH
Department of Pharmacology, Medical School, The University of Michigan, Ann Arbor, Michigan 48109-0606, USA.
Vatsis
K P
KP
Weber
W W
WW
D020413:3' Untranslated Regions / Q000235:genetics*; D000123:Acetyltransferases / Q000235:genetics* / Q000378:metabolism; D000483:Alleles; D000818:Animals; D001191:Arylamine N-Acetyltransferase; D019556:COS Cells; D004926:Escherichia coli; D005260:Female; D015870:Gene Expression; D005838:Genotype; D006801:Humans; D007527:Isoenzymes; D007962:Leukocytes; D008297:Male; D009154:Mutation; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D016364:Reading Frames; D011994:Recombinant Proteins / Q000378:metabolism
D020413:3' Untranslated Regions; D007527:Isoenzymes; D012322:RNA Precursors; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000123:Acetyltransferases; D001191:Arylamine N-Acetyltransferase; C089384:N-acetyltransferase 1