Mapping the Initial Stages of a Protective Pathway that Enhances Catalytic Turnover by a Lytic Polysaccharide Monooxygenase.
Mapping the Initial Stages of a Protective Pathway that Enhances Catalytic Turnover by a Lytic Polysaccharide Monooxygenase.
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DOI：
10.1021/jacs.3c06607
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发表时间：
2023-09-20
影响因子：
15
通讯作者：
Green, Anthony P.
中科院分区：
文献类型：
--
作者：
Zhao, Jingming;Zhuo, Ying;Diaz, Daniel E.;Shanmugam, Muralidharan;Telfer, Abbey J.;Lindley, Peter J.;Kracher, Daniel;Hayashi, Takahiro;Seibt, Lisa S.;Hardy, Florence J.;Manners, Oliver;Hedison, Tobias M.;Hollywood, Katherine A.;Spiess, Reynard;Cain, Kathleen M.;Diaz-Moreno, Sofia;Scrutton, Nigel S.;Tovborg, Morten;Walton, Paul H.;Heyes, Derren J.;Green, Anthony P.
加氧酶和过氧化物酶在其活性部位产生中间体，从而控制底物中惰性C-H键的官能化，例如在甲烷酶促转化为甲醇的过程中。然而，为了成为可行的催化剂，这些酶还必须防止对基本活性中心残基的氧化损伤，这可能发生在偶联和非偶联周转过程中。在这里，我们使用停流光谱、靶向突变、TD-DFT计算、高能分辨荧光检测X射线吸收光谱和电子顺磁共振光谱相结合的方法来研究两个瞬时中间体，它们共同构成了铜依赖的裂解多糖单加氧酶(LPMOS)活性部位的保护途径。首先，在没有底物的情况下，用过氧化氢或过氧酸处理LPMO后，在铜组氨酸支撑活性中心产生了一个瞬时的高价物种。这种中间体，我们认为是CuII-(组氨酸自由基)，然后在跨系统反应中与附近的酪氨酸残基反应，生成铁磁性耦合的(S=1)CuII-酪氨酸自由基对，从而将组氨酸支撑活性中心恢复到其静止状态，并允许它通过还原重新进入催化循环。这一过程使酶有能力将对活性部位组氨酸残基的损害降至最低，从而在酶失活之前增加总周转次数，突显氧化酶是如何进化的，以保护自己在非偶联周转期间免受有害副反应的影响。
Oxygenase and peroxygenase enzymes generate intermediates at their active sites which bring about the controlled functionalization of inert C–H bonds in substrates, such as in the enzymatic conversion of methane to methanol. To be viable catalysts, however, these enzymes must also prevent oxidative damage to essential active site residues, which can occur during both coupled and uncoupled turnover. Herein, we use a combination of stopped-flow spectroscopy, targeted mutagenesis, TD-DFT calculations, high-energy resolution fluorescence detection X-ray absorption spectroscopy, and electron paramagnetic resonance spectroscopy to study two transient intermediates that together form a protective pathway built into the active sites of copper-dependent lytic polysaccharide monooxygenases (LPMOs). First, a transient high-valent species is generated at the copper histidine brace active site following treatment of the LPMO with either hydrogen peroxide or peroxyacids in the absence of substrate. This intermediate, which we propose to be a CuII–(histidyl radical), then reacts with a nearby tyrosine residue in an intersystem-crossing reaction to give a ferromagnetically coupled (S = 1) CuII–tyrosyl radical pair, thereby restoring the histidine brace active site to its resting state and allowing it to re-enter the catalytic cycle through reduction. This process gives the enzyme the capacity to minimize damage to the active site histidine residues “on the fly” to increase the total turnover number prior to enzyme deactivation, highlighting how oxidative enzymes are evolved to protect themselves from deleterious side reactions during uncoupled turnover.
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影响因子：
3.6
作者：
Das, Arpan;Ahmed, Jasimuddin;Mandal, Swadhin K.
通讯作者：
Mandal, Swadhin K.
影响因子：
15
作者：
KAU, LS;SPIRASOLOMON, DJ;SOLOMON, EI
通讯作者：
SOLOMON, EI
影响因子：
15
作者：
Gao, Jinshan;Thomas, Daniel A.;Beauchamp, J. L.
通讯作者：
Beauchamp, J. L.
DOI：
10.1039/a903403j
发表时间：
1999-01-01
期刊：
JOURNAL OF THE CHEMICAL SOCIETY-PERKIN TRANSACTIONS 2
影响因子：
--
作者：
Gilbert, BC;Silvester, S;Whitwood, AC
通讯作者：
Whitwood, AC
影响因子：
15
作者：
Kim, Sunghee;Ginsbach, Jake W.;Lee, Jung Yoon;Peterson, Ryan L.;Liu, Jeffrey J.;Siegler, Maxime A.;Sarjeant, Amy A.;Solomon, Edward I.;Karlin, Kenneth D.
通讯作者：
Karlin, Kenneth D.