Acebes Sandra, Ruiz-Dueñas Francisco J, Toubes Mario, Sáez-Jiménez Veronica, Pérez-Boada Marta, Lucas M Fátima, Martínez Angel T, Guallar Victor
Barcelona Supercomputing Center, Joint BSC-CRG-IRB Research Program in Computational Biology , Jordi Girona 29, E-08034 Barcelona, Spain.
Centro de Investigaciones Biológicas, CSIC , Ramiro de Maeztu 9, E-28040 Madrid, Spain.
J Phys Chem B. 2017 Apr 27;121(16):3946-3954. doi: 10.1021/acs.jpcb.7b00835. Epub 2017 Apr 14.
Combining a computational analysis with site-directed mutagenesis, we have studied the long-range electron transfer pathway in versatile and lignin peroxidases, two enzymes of biotechnological interest that play a key role for fungal degradation of the bulky lignin molecule in plant biomass. The in silico study established two possible electron transfer routes starting at the surface tryptophan residue previously identified as responsible for oxidation of the bulky lignin polymer. Moreover, in both enzymes, a second buried tryptophan residue appears as a top electron transfer carrier, indicating the prevalence of one pathway. Site-directed mutagenesis of versatile peroxidase (from Pleurotus eryngii) allowed us to corroborate the computational analysis and the role played by the buried tryptophan (Trp244) and a neighbor phenylalanine residue (Phe198), together with the surface tryptophan, in the electron transfer. These three aromatic residues are highly conserved in all the sequences analyzed (up to a total of 169). The importance of the surface (Trp171) and buried (Trp251) tryptophan residues in lignin peroxidase has been also confirmed by directed mutagenesis of the Phanerochaete chrysosporium enzyme. Overall, the combined procedure identifies analogous electron transfer pathways in the long-range oxidation mechanism for both ligninolytic peroxidases, constituting a good example of how computational analysis avoids making extensive trial-error mutagenic experiments.
通过将计算分析与定点诱变相结合，我们研究了多功能过氧化物酶和木质素过氧化物酶中的远程电子转移途径，这两种酶在生物技术领域具有重要意义，在真菌降解植物生物质中庞大的木质素分子过程中发挥关键作用。计算机模拟研究确定了两条可能的电子转移途径，起始于先前被确定为负责氧化庞大木质素聚合物的表面色氨酸残基。此外，在这两种酶中，第二个埋藏的色氨酸残基似乎是主要的电子转移载体，表明存在一条优势途径。对来自杏鲍菇的多功能过氧化物酶进行定点诱变，使我们能够证实计算分析以及埋藏的色氨酸（Trp244）和相邻苯丙氨酸残基（Phe198）与表面色氨酸在电子转移中所起的作用。这三个芳香族残基在所有分析的序列中（总共169个）高度保守。通过对黄孢原毛平革菌酶进行定点诱变，也证实了木质素过氧化物酶中表面（Trp171）和埋藏（Trp251）色氨酸残基的重要性。总体而言，这种组合方法确定了两种木质素分解过氧化物酶在远程氧化机制中的类似电子转移途径，这是计算分析如何避免进行大量试错诱变实验的一个很好的例子。
