In vitro functional analysis of human cytochrome P450 2A13 genetic variants: P450 2A13*2,*3,*4, and*10
In vitro functional analysis of human cytochrome P450 2A13 genetic variants: P450 2A13*2,*3,*4, and*10
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DOI：
10.1080/15287394.2018.1460784
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发表时间：
2018-01-01
影响因子：
2.6
通讯作者：
Kim, Donghak
中科院分区：
文献类型：
--
作者：
Kim, Vitchan;Yeom, Sora;Kim, Donghak
人类在2A亚家族中有三种细胞色素P450酶(2A6、2A7和2A13)。P450 2A13主要在人的气管和肺中表达，而P450 2A6在人的肝脏中有表达。P450 2A13酶可能是许多烟草特有致癌物代谢激活的主要酶。基因变异显著影响吸烟所导致的毒理学后果。本研究旨在检测P450 2A13*2、*3、*4和*10等位基因中5个P450 2A13基因变异(R257C、133_134insT、R101Q、I331T和R257C/I331T)的体外功能活性。构建了突变克隆，并在大肠杆菌中表达了重组酶。含有R257C、133_134insT、I331T和R257C/I331T的P450 2A13突变体表现出P450全酶谱。突变体R101Q未见明显表达。P450 2A13酶与香豆素呈现典型的I型结合谱，R257C、R257C/I331T和133_134insT突变体的计算结合亲和力降低约3-7倍。在纯化的突变酶对香豆素和尼古丁的催化分析中，R257C和I331T突变株表现出较低的k(CAT)值，催化效率降低了约20%。R257C/I331T双突变导致K-m值升高，k(CAT)值降低，导致催化效率下降50%。对于133_134insT突变体，酶活性没有明显饱和，但在最高浓度下测得的催化活性显著低于野生型或其他突变型酶。对P4502A13等位基因变异的功能分析可能有助于理解P4502A13多态在许多烟草来源致癌物生物活性中的后果。
Humans possess three cytochrome P450 enzymes in the 2A subfamily (2A6, 2A7, and 2A13). P450 2A13 is mainly expressed in the human trachea and lung, whereas P450 2A6 is found in human liver. The P450 2A13 enzyme may be considered as the primary enzyme responsible for metabolic activation of many tobacco-specific carcinogens. Genetic variations significantly influence the toxicological consequences attributed to tobacco smoking. The aim of this study was to examine the in vitro functional activities of five P450 2A13 genetic variations (R257C, 133_134insT, R101Q, I331T, and R257C/I331T) in P450 2A13*2, *3, *4, and *10 alleles. Mutant clones were constructed and their recombinant enzymes were expressed in Escherichia coli. P450 2A13 mutants containing R257C, 133_134insT, I331T, and R257C/I331T displayed P450 holoenzyme spectra. The R101Q mutant was not apparently expressed. P450 2A13 enzymes displayed the typical type I binding spectra to coumarin and the calculated binding affinities of R257C, R257C/I331T, and 133_134insT mutants were decreased approximately three- to sevenfold. In catalytic analyses of purified mutant enzymes for coumarin and nicotine, the R257C and I331T mutants exhibited lower k(cat) values with catalytic efficiencies reduced up to approximately 20%. The double mutation of R257C/I331T induced increased K-m values and diminished k(cat) values that resulted in >50% decrease in catalytic efficiencies. For 133_134insT mutant, catalytic activities were not markedly saturated but the measured rates at the highest concentrations were significantly lower than those of the wild-type or other mutant enzymes. Functional analysis of these variations in P450 2A13 allelic variants may help to understand the consequences of P450 2A13 polymorphism in bioactivation of many tobacco-derived carcinogens.