Institute of Biomedical Sciences, Academia Sinica, Nankang, Taipei, Taiwan, Republic of China.
PLoS One. 2011;6(9):e25801. doi: 10.1371/journal.pone.0025801. Epub 2011 Sep 29.
Arylamine N-acetyltransferase 2 (NAT2) is an important catalytic enzyme that metabolizes the carcinogenic arylamines, hydrazine drugs and chemicals. This enzyme is highly polymorphic in different human populations. Several polymorphisms of NAT2, including the single amino acid substitutions R64Q, I114T, D122N, L137F, Q145P, R197Q, and G286E, are classified as slow acetylators, whereas the wild-type NAT2 is classified as a fast acetylator. The slow acetylators are often associated with drug toxicity and efficacy as well as cancer susceptibility. The biological functions of these 7 mutations have previously been characterized, but the structural basis behind the reduced catalytic activity and reduced protein level is not clear.
METHODOLOGY/PRINCIPAL FINDINGS: We performed multiple molecular dynamics simulations of these mutants as well as NAT2 to investigate the structural and dynamical effects throughout the protein structure, specifically the catalytic triad, cofactor binding site, and the substrate binding pocket. None of these mutations induced unfolding; instead, their effects were confined to the inter-domain, domain 3 and 17-residue insert region, where the flexibility was significantly reduced relative to the wild-type. Structural effects of these mutations propagate through space and cause a change in catalytic triad conformation, cofactor binding site, substrate binding pocket size/shape and electrostatic potential.
CONCLUSIONS/SIGNIFICANCE: Our results showed that the dynamical properties of all the mutant structures, especially in inter-domain, domain 3 and 17-residue insert region were affected in the same manner. Similarly, the electrostatic potential of all the mutants were altered and also the functionally important regions such as catalytic triad, cofactor binding site, and substrate binding pocket adopted different orientation and/or conformation relative to the wild-type that may affect the functions of the mutants. Overall, our study may provide the structural basis for reduced catalytic activity and protein level, as was experimentally observed for these polymorphisms.
芳香胺 N-乙酰基转移酶 2（NAT2）是一种重要的催化酶，可代谢致癌芳香胺、肼类药物和化学物质。该酶在不同人群中高度多态。NAT2 的几种多态性，包括单一氨基酸取代 R64Q、I114T、D122N、L137F、Q145P、R197Q 和 G286E，被归类为慢乙酰化酶，而野生型 NAT2 被归类为快乙酰化酶。慢乙酰化酶通常与药物毒性和疗效以及癌症易感性有关。这些 7 种突变的生物学功能以前已经得到了描述，但降低催化活性和降低蛋白质水平的结构基础尚不清楚。
方法/主要发现：我们对这些突变体以及 NAT2 进行了多次分子动力学模拟，以研究整个蛋白质结构，特别是催化三联体、辅因子结合位点和底物结合口袋的结构和动力学效应。这些突变没有引起蛋白质的解折叠，而是将其影响局限在结构域之间、结构域 3 和 17 个残基插入区域，与野生型相比，这些区域的灵活性明显降低。这些突变的结构效应通过空间传播，导致催化三联体构象、辅因子结合位点、底物结合口袋大小/形状和静电势发生变化。
结论/意义：我们的结果表明，所有突变体结构的动力学特性，特别是在结构域之间、结构域 3 和 17 个残基插入区域，受到了同样的影响。同样，所有突变体的静电势都发生了改变，而且功能重要的区域，如催化三联体、辅因子结合位点和底物结合口袋，相对于野生型，采取了不同的取向和/或构象，这可能影响突变体的功能。总的来说，我们的研究可能为这些多态性实验观察到的降低催化活性和蛋白质水平提供了结构基础。
