Human acetyl CoA:arylamine N-acetyltransferase variants generated by random mutagenesis.
Human acetyl CoA:arylamine N-acetyltransferase variants generated by random mutagenesis.
复制标题
通过随机诱变产生的人乙酰辅酶 A：芳基胺 N-乙酰转移酶变体。
作者：
Joanna Summerscales;P. Josephy
乙酰辅酶A：芳胺N-乙酰基转移酶(NAT)催化芳香胺的N-乙酰化和芳基羟胺的O-乙酰化，这些反应控制着许多药物和致癌物的处置和毒性。人类NAT基因和酶NA1和NAT2是高度多态的，构成了药物代谢遗传控制的最好研究例子之一。自然产生的人类NAT变体对NAT氨基酸序列和酶活性之间的关系提供了有限的洞察力。我们以前已经证明，重组Nat2在细菌测试菌株中的表达导致对诱变的硝基芳香族化合物(通过细菌酶还原为芳香胺)的敏感度大大提高。我们假设随机突变和快速筛选相结合可以用于鉴定NAT酶中具有重要功能的氨基酸残基。通过聚合酶链式反应介导的完全编码序列的随机突变来产生Nat2变异体库。依赖NAT的诱变剂3-甲基-2-硝基咪唑[4，5-f]喹啉诱导的逆转被用作筛选这些库的基础，以鉴定酶活性改变的变异体。对18个突变体进行了定量致突变性分析和酶动力学测定。这种方法可以为参与诱变剂代谢激活的酶的生物化学提供新的见解。
Acetyl CoA:arylamine N-acetyltransferase (NAT) enzymes catalyze the N-acetylation of aromatic amines and the O-acetylation of aryl hydroxylamines, reactions that govern the disposition and toxicity of many drugs and carcinogens. The human NAT genes and enzymes NAT1 and NAT2 are highly polymorphic and constitute one of the best studied examples of the genetic control of drug metabolism. Naturally occurring human NAT variants provide limited insight into the relationship between NAT amino acid sequence and enzyme activity. We have shown previously that the expression of recombinant NAT2 in bacterial tester strains results in greatly enhanced sensitivity to mutagenic nitroaromatic compounds (which are reduced to aryl hydroxylamines by bacterial enzymes). We hypothesized that random mutagenesis combined with rapid screening could be used to identify functionally significant amino acid residues in NAT enzymes. Pools of NAT2 variants were generated by polymerase chain reaction-mediated random mutagenesis of the complete coding sequence. Reversion induced by a NAT-dependent mutagen, 3-methyl-2-nitroimidazo[4,5-f]quinoline, was used as the basis for screening these pools to identify variants with altered enzyme activity. Eighteen variants were characterized by quantitative mutagenicity assays and enzyme kinetic measurements. This approach can provide new insight into the biochemistry of enzymes involved in the metabolic activation of mutagens.
DOI：
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发表时间：
1998-05
期刊：
Drug metabolism and disposition: the biological fate of chemicals
影响因子：
--
作者：
L. Estrada-Rodgers;G. Levy;W. Weber
通讯作者：
L. Estrada-Rodgers;G. Levy;W. Weber
影响因子：
3.9
作者：
Nakamura, K;Martin, MV;Guengerich, FP
通讯作者：
Guengerich, FP
创建对 O6-苄基鸟嘌呤具有抗性的人烷基转移酶。
影响因子：
11.2
作者：
Christians,FC;Dawson,BJ;Coates,MM;Loeb,LA
通讯作者：
Loeb,LA