Shoji Mitsuo, Murakawa Takeshi, Nakanishi Shota, Boero Mauro, Shigeta Yasuteru, Hayashi Hideyuki, Okajima Toshihide
Center for Computational Sciences, University of Tsukuba 1-1-1 Tennodai Tsukuba 305-8577 Ibaraki Japan
JST-PRESTO 4-1-8 Honcho Kawaguchi 332-0012 Saitama Japan.
Chem Sci. 2022 Aug 23;13(36):10923-10938. doi: 10.1039/d2sc01356h. eCollection 2022 Sep 21.
Copper amine oxidase from (AGAO) catalyses the oxidative deamination of primary amines a large conformational change of a topaquinone (TPQ) cofactor during the semiquinone formation step. This conformational change of TPQ occurs in the presence of strong hydrogen bonds and neighboring bulky amino acids, especially the conserved Asn381, which restricts TPQ conformational changes over the catalytic cycle. Whether such a semiquinone intermediate is catalytically active or inert has been a matter of debate in copper amine oxidases. Here, we show that the reaction rate of the Asn381Ala mutant decreases 160-fold, and the X-ray crystal structures of the mutant reveals a TPQ-flipped conformation in both the oxidized and reduced states, preceding semiquinone formation. Our hybrid quantum mechanics/molecular mechanics (QM/MM) simulations show that the TPQ conformational change is realized through the sequential steps of the TPQ ring-rotation and slide. We determine that the bulky side chain of Asn381 hinders the undesired TPQ ring-rotation in the oxidized form, favoring the TPQ ring-rotation in reduced TPQ by a further stabilization leading to the TPQ semiquinone form. The acquired conformational flexibility of TPQ semiquinone promotes a high reactivity of Cu(i) to O, suggesting that the semiquinone form is catalytically active for the subsequent oxidative half-reaction in AGAO. The ingenious molecular mechanism exerted by TPQ to achieve the "state-specific" reaction sheds new light on a drastic environmental transformation around the catalytic center.
来自[具体来源未给出]的铜胺氧化酶（AGAO）催化伯胺的氧化脱氨反应，在半醌形成步骤中，对苯二酚醌（TPQ）辅因子会发生较大的构象变化。TPQ的这种构象变化发生在存在强氢键和相邻大体积氨基酸的情况下，尤其是保守的Asn381，它在催化循环中限制了TPQ的构象变化。在铜胺氧化酶中，这种半醌中间体是具有催化活性还是惰性一直存在争议。在这里，我们表明Asn381Ala突变体的反应速率降低了160倍，并且该突变体的X射线晶体结构显示在半醌形成之前，氧化态和还原态的TPQ均呈翻转构象。我们的量子力学/分子力学（QM/MM）混合模拟表明，TPQ的构象变化是通过TPQ环旋转和滑动的连续步骤实现的。我们确定Asn381的大体积侧链在氧化形式下阻碍了不期望的TPQ环旋转，通过进一步稳定作用有利于还原态TPQ中的TPQ环旋转，从而导致TPQ半醌形式。TPQ半醌获得的构象灵活性促进了Cu(i)对O的高反应性，这表明半醌形式对AGAO中随后的氧化半反应具有催化活性。TPQ发挥作用以实现“状态特异性”反应的巧妙分子机制为催化中心周围剧烈的环境转变提供了新的线索。
