Crystal structures of pristine and oxidatively processed lignin peroxidase expressed in Escherichia coli and of the W171F variant that eliminates the redox active tryptophan 171.: Implications for the reaction mechanism
Crystal structures of pristine and oxidatively processed lignin peroxidase expressed in Escherichia coli and of the W171F variant that eliminates the redox active tryptophan 171.: Implications for the reaction mechanism
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DOI：
10.1006/jmbi.2000.4346
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发表时间：
2001-01-26
影响因子：
5.6
通讯作者：
Piontek, K
中科院分区：
文献类型：
--
作者：
Blodig, W;Smith, AT;Piontek, K
来自白腐菌Phanerochaete chrysosporium的血红素酶木质素过氧化物酶(LIP)含有一个暴露在溶剂中的氧化还原活性色氨酸残基(Trp171)，它携带一个独特的羟基，立体地连接到它的C-β原子上。Trp171Phe突变体对底物藜芦醇没有任何活性。涉及β-羟基-Trp171的藜芦醇氧化机理很大程度上是未知的。在这里，我们提出了高分辨率的LIP同工酶H8的第一个晶体结构，其原始的非羟化形式，C-β-羟化的形式，以及Trp171Phe突变体的晶体结构，使用重组表达和复性的蛋白从大肠杆菌产生。因此，所有的结构都是无糖化的。对突变的反应结构变化是边缘的，并允许我们将完全缺乏活性完全归因于氧化还原活性吲哚侧链的缺乏。Trp171羟基化的立体特异性的起源可以从结构上解释。提出了Trp171参与的反应机理，并讨论了该修饰的可能作用。关于正在进行的关于休眠状态下血红素铁的配位状态的辩论的另一个重要结果是，在所有情况下，铁都是六个坐标，数据是在室温下收集的。铁到远端水配体的平均距离为2.18(+/-0.08)埃。在室温下的数据收集过程中，发现自由基清除剂地衣酚可以减少对晶体的辐射损伤。(C)2001年学术出版社。
The heme enzyme lignin peroxidase (LiP) from the white rot fungus Phanerochaete chrysosporium contains a solvent exposed redox active tryptophan residue (Trp171) that carries a unique hydroxy group stereospecifically attached to its C-beta atom. A Trp171Phe mutant has no activity at all towards the substrate veratryl alcohol. The mechanism of veratryl alcohol oxidation involving beta -hydroxy-Trp171 is largely unknown. Here, we present the first crystal structures of LiP isozyme H8 at high resolution in its pristine non-hydroxylated form, of the C-beta-hydroxylated form, and of the Trp171Phe mutant using recombinantly expressed and refolded protein produced from Escherichia coli. As a consequence, all structures are unglycosylated. Structural changes in response to the mutation are marginal and allow us to attribute the complete lack of activity exclusively to the absence of the redox active indole side-chain. The origin of the stereospecificity of the Trp171 hydroxylation can be explained on structural grounds. A reaction mechanism involving Trp171 is proposed and the possible function of the modification is discussed. Another important result regarding the ongoing debate on the co-ordination state of the heme iron in the resting state is that the iron is six coordinate in all cases the data being collected at room temperature. The mean distance from the iron to the distal water Ligand is 2.18(+/-0.08) Angstrom. The radical scavenger orcinol was found to decrease radiation damage to the crystals, during data collection at room temperature. (C) 2001 Academic Press.