Oxygen Activation Switch in the Copper Amine Oxidase of Escherichia coli.
Oxygen Activation Switch in the Copper Amine Oxidase of Escherichia coli.
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DOI：
10.1021/acs.biochem.8b00633
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发表时间：
2018-09-11
期刊：
影响因子：
2.9
通讯作者：
McPherson MJ
中科院分区：
文献类型：
--
作者：
Gaule TG;Smith MA;Tych KM;Pirrat P;Trinh CH;Pearson AR;Knowles PF;McPherson MJ
铜胺氧化酶(CuAOs)是一种在伯胺催化周转过程中将分子氧还原为过氧化氢的金属酶。除了活性中心的Cu2+外，活性中心的两个外周钙中心∼32？在大肠杆菌胺氧化酶中也有作用。埋藏的钙离子(Asp533、Leu534、Asp535、Asp678和Ala679)是全长蛋白质生产所必需的，而表面钙离子(Glu573、Tyr667、Asp670和Glu672)调节2，4，5-三羟基苯丙氨酸醌(TPQ)辅因子的生物发生。表面位置的E573Q突变阻止了钙结合和TPQ的生物发生。然而，TPQ的生物发生可以通过抑制突变(I342F)来恢复。I342F/E573Q在支持TPQ生物合成(∼60%WTECAO TPQ)的同时，几乎没有胺氧化酶活性(∼4.6%WTECAO活性)。为了了解这些远程突变是如何对TPQ的生物发生和催化产生重大影响的，我们使用了紫外-可见光谱、稳态动力学、抑制试验和X射线结晶学。结果表明，表面金属位控制着Cu2+-底物还原TPQ(TPQAMQ)-Cu2+-TPQSQ(TPQSQ)对的平衡(歧化)。通过络合或诱变来去除这个位点上的钙离子，将平衡转移到Cu2+-TPQAMQ或破坏了Cu2+-TPQSQ的稳定。晶体结构分析表明，在Cu2+-酪氨酸状态下，TPQ的生物合成在去质子化时停滞。我们的发现支持WTECAO在催化过程中使用内球电子转移机制进行氧还原，虽然铜+-酪氨酸自由基中间体对TPQ的生物发生不是必需的，但它是有效的生物发生所必需的。
Copper amine oxidases (CuAOs) are metalloenzymes that reduce molecular oxygen to hydrogen peroxide during catalytic turnover of primary amines. In addition to Cu2+ in the active site, two peripheral calcium sites, ∼32 Å from the active site, have roles in Escherichia coli amine oxidase (ECAO). The buried Ca2+ (Asp533, Leu534, Asp535, Asp678, and Ala679) is essential for full-length protein production, while the surface Ca2+ (Glu573, Tyr667, Asp670, and Glu672) modulates biogenesis of the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor. The E573Q mutation at the surface site prevents calcium binding and TPQ biogenesis. However, TPQ biogenesis can be restored by a suppressor mutation (I342F) in the proposed oxygen delivery channel to the active site. While supporting TPQ biogenesis (∼60% WTECAO TPQ), I342F/E573Q has almost no amine oxidase activity (∼4.6% WTECAO activity). To understand how these long-range mutations have major effects on TPQ biogenesis and catalysis, we employed ultraviolet–visible spectroscopy, steady-state kinetics, inhibition assays, and X-ray crystallography. We show that the surface metal site controls the equilibrium (disproportionation) of the Cu2+-substrate reduced TPQ (TPQAMQ) Cu+-TPQ semiquinone (TPQSQ) couple. Removal of the calcium ion from this site by chelation or mutagenesis shifts the equilibrium to Cu2+-TPQAMQ or destabilizes Cu+-TPQSQ. Crystal structure analysis shows that TPQ biogenesis is stalled at deprotonation in the Cu2+-tyrosinate state. Our findings support WTECAO using the inner sphere electron transfer mechanism for oxygen reduction during catalysis, and while a Cu+-tyrosyl radical intermediate is not essential for TPQ biogenesis, it is required for efficient biogenesis.
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影响因子：
4.8
作者：
Evans, JP;Ahn, K;Klinman, JP
通讯作者：
Klinman, JP
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
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通讯作者：
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影响因子：
2.9
作者：
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通讯作者：
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影响因子：
3.2
作者：
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通讯作者：
McPherson, Michael J.
影响因子：
3
作者：
Dooley, DM;Brown, DE
通讯作者：
Brown, DE