Substrate oxidation sites in versatile peroxidase and other basidiomycete peroxidases
Substrate oxidation sites in versatile peroxidase and other basidiomycete peroxidases
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DOI：
10.1093/jxb/ern261
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发表时间：
2009-02-01
影响因子：
6.9
通讯作者：
Martinez, Angel T.
中科院分区：
文献类型：
--
作者：
Ruiz-Duenas, Francisco J.;Morales, Maria;Martinez, Angel T.
多功能过氧化物酶（VP）是通过其氧化其他担子菌过氧化物酶的典型底物的能力来定义的：(i) Mn2+，锰过氧化物酶（MnP）底物（Mn3+能够氧化酚并引发脂质过氧化反应）；（ii）木质素过氧化物酶（LiP）的典型底物戊四醇（VA）；（iii）简单酚类物质，它们是Coprinopsis cinerea过氧化物酶（CIP）的底物。晶体学、光谱学、定向诱变和动力学研究表明，这些“杂交”特性是由于在一个蛋白质中存在不同的催化位点，使人想起其他担子菌过氧化物酶家族。与MnP相比，野生VP和重组VP的晶体结构以及突变变异体的动力学显示其mn氧化位点存在一定差异。这导致在形成其结合位点的三个酸性残基中只有两个存在的情况下，Mn2+有效氧化。另一方面，溶剂暴露的色氨酸是VA氧化中的催化活性残留物，启动电子传递途径到血红素（其他两个假定的途径被诱变抛弃）。用电子顺磁共振检测过氧化氢活化VP后色氨酸自由基的形成。这是第一次在木质素分解过氧化物酶中直接发现蛋白质自由基。与LiP相反，VP催化色氨酸在过氧化氢过量下不会被羟基化。色氨酸环境也影响了催化作用，它的修饰引入了VP的一些LiP特性。此外，一些酚类和染料在血红素通道的边缘被VP氧化，就像在CIP中发现的那样。最后，讨论了副产物的生物技术意义。
Versatile peroxidase (VP) is defined by its capabilities to oxidize the typical substrates of other basidiomycete peroxidases: (i) Mn2+, the manganese peroxidase (MnP) substrate (Mn3+ being able to oxidize phenols and initiate lipid peroxidation reactions); (ii) veratryl alcohol (VA), the typical lignin peroxidase (LiP) substrate; and (iii) simple phenols, which are the substrates of Coprinopsis cinerea peroxidase (CIP). Crystallographic, spectroscopic, directed mutagenesis, and kinetic studies showed that these 'hybrid' properties are due to the coexistence in a single protein of different catalytic sites reminiscent of those present in the other basidiomycete peroxidase families. Crystal structures of wild and recombinant VP, and kinetics of mutated variants, revealed certain differences in its Mn-oxidation site compared with MnP. These result in efficient Mn2+ oxidation in the presence of only two of the three acidic residues forming its binding site. On the other hand, a solvent-exposed tryptophan is the catalytically-active residue in VA oxidation, initiating an electron transfer pathway to haem (two other putative pathways were discarded by mutagenesis). Formation of a tryptophanyl radical after VP activation by peroxide was detected using electron paramagnetic resonance. This was the first time that a protein radical was directly demonstrated in a ligninolytic peroxidase. In contrast with LiP, the VP catalytic tryptophan is not beta-hydroxylated under hydrogen peroxide excess. It was also shown that the tryptophan environment affected catalysis, its modification introducing some LiP properties in VP. Moreover, some phenols and dyes are oxidized by VP at the edge of the main haem access channel, as found in CIP. Finally, the biotechnological interest of VP is discussed.