Abstract:
:Acetyl CoA:arylamine N-acetyltransferase (NAT; E.C. 2.3.1.5) enzymes play a key role in the metabolic activation of aromatic amine and nitroaromatic mutagens to electrophilic reactive intermediates. We have developed a system in which the activation of mutagens by recombinant human NAT2, expressed in Escherichia coli, can be detected by the appearance of Lac+ revertants. The mutagenesis assay is based on the reversion of an E. coli lacZ frameshift allele; the host strain for the assay is devoid of endogenous NAT activity and a plasmid vector is used for expression of human NAT2. A high-throughput version of the assay facilitates rapid screening of pools of NAT2 variants generated (for example) by random mutagenesis. Along with the methods for these assays, we present selected results of a screening effort in which mutations along the length of the NAT2 sequence have been examined. Homology modeling and simulated annealing have been used to analyze the potential effects of these mutations on structural integrity and substrate binding.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Savulescu MR,Mushtaq A,Josephy PDdoi
10.1016/S0076-6879(05)00011-Xsubject
Has Abstractpub_date
2005-01-01 00:00:00pages
192-215eissn
0076-6879issn
1557-7988pii
S0076-6879(05)00011-Xjournal_volume
400pub_type
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