Formation of a tyrosine adduct involved in lignin degradation by Trametopsis cervina lignin peroxidase: a novel peroxidase activation mechanism
Formation of a tyrosine adduct involved in lignin degradation by Trametopsis cervina lignin peroxidase: a novel peroxidase activation mechanism
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DOI：
10.1042/bj20130251
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发表时间：
2013-06-15
影响因子：
4.1
通讯作者：
Martinez, Angel T.
中科院分区：
文献类型：
--
作者：
Miki, Yuta;Pogni, Rebecca;Martinez, Angel T.
来自Trametopsis cerebral的LiP（木质素过氧化物酶）具有暴露的催化酪氨酸残基（Tyr（181）），而不是其它木质素降解过氧化物酶中保守的色氨酸。原始LiP在VA（藜芦醇）氧化中显示滞后期。然而，VA-LiP（用H2 O2和VA处理后的LiP）没有这种滞后，并且H2 O2-LiP（H2 O2处理的LiP）是无活性的。MS分析表明，VA-LiP包括一个与Tyr侧链共价结合的VA分子（181），而H2 O2-LiP含有羟基化Tyr（181）。在Y171 N变体中没有形成加合物。分子对接显示，VA结合通过与Tyr（181）和Phe（89）的夹心式pi堆叠而得到促进。EPR光谱过氧化物活化后的预处理的LiP显示蛋白质自由基以外的酪氨酸自由基中发现的原始LiP，这被分配到一个酪氨酸VA加合物自由基在VA-LiP和二羟基苯丙氨酸自由基在H2 O2-LiP。这两种自由基都能够氧化大的低氧化还原电位底物，但H2 O2-LiP无法氧化高氧化还原电位底物。瞬态动力学表明，酪氨酸VA加合物强烈促进（>100倍）底物氧化的化合物LE，在催化的限速步骤。新的活化机制参与木质素分解，如使用木质素模型底物所示。本文是第一次报告的自催化修饰，导致功能的改变，第二类过氧化物酶。
LiP (lignin peroxidase) from Trametopsis cervina has an exposed catalytic tyrosine residue (Tyr(181)) instead of the tryptophan conserved in other lignin-degrading peroxidases. Pristine LiP showed a lag period in VA (veratryl alcohol) oxidation. However, VA-LiP (LiP after treatment with H2O2 and VA) lacked this lag, and H2O2-LiP (H2O2-treated LiP) was inactive. MS analyses revealed that VA-LiP includes one VA molecule covalently bound to the side chain of Tyr(181), whereas H2O2-LiP contains a hydroxylated Tyr(181). No adduct is formed in the Y171N variant. Molecular docking showed that VA binding is favoured by sandwich pi stacking with Tyr(181) and Phe(89). EPR spectroscopy after peroxide activation of the pre-treated LiPs showed protein radicals other than the tyrosine radical found in pristine LiP, which were assigned to a tyrosine VA adduct radical in VA-LiP and a dihydroxyphenyalanine radical in H2O2-LiP. Both radicals are able to oxidize large low-redox-potential substrates, but H2O2-LiP is unable to oxidize high-redox-potential substrates. Transient-state kinetics showed that the tyrosine VA adduct strongly promotes (>100-fold) substrate oxidation by compound LE, the rate-limiting step in catalysis. The novel activation mechanism is involved in ligninolysis, as demonstrated using lignin model substrates. The present paper is the first report on autocatalytic modification, resulting in functional alteration, among class II peroxidases.