Tsukihara Takahisa, Honda Yoichi, Sakai Ryota, Watanabe Takahito, Watanabe Takashi
Research Institute for Sustainable Humanosphere, Kyoto University, Gokasho Uji, Kyoto 611-0011, Japan.
Appl Environ Microbiol. 2008 May;74(9):2873-81. doi: 10.1128/AEM.02080-07. Epub 2008 Mar 7.
Unlike general peroxidases, Pleurotus ostreatus MnP2 was reported to have a unique property of direct oxidization of high-molecular-weight compounds, such as Poly R-478 and RNase A. To elucidate the mechanism for oxidation of polymeric substrates by MnP2, a series of mutant enzymes were produced by using a homologous gene expression system, and their reactivities were characterized. A mutant enzyme with an Ala substituting for an exposing Trp (W170A) drastically lost oxidation activity for veratryl alcohol (VA), Poly R-478, and RNase A, whereas the kinetic properties for Mn(2+) and H(2)O(2) were substantially unchanged. These results demonstrated that, in addition to VA, the high-molecular-weight substrates are directly oxidized by MnP2 at W170. Moreover, in the mutants Q266F and V166/168L, amino acid substitution(s) around W170 resulted in a decreased activity only for the high-molecular-weight substrates. These results, along with the three-dimensional modeling of the mutants, suggested that the mutations caused a steric hindrance to access of the polymeric substrates to W170. Another mutant, R263N, contained a newly generated N glycosylation site and showed a higher molecular mass in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Interestingly, the R263N mutant exhibited an increased reactivity with VA and high-molecular-weight substrates. The existence of an additional carbohydrate modification and the catalytic properties in this mutant are discussed. This is the first study of a direct mechanism for oxidation of high-molecular-weight substrates by a fungal peroxidase using a homologous gene expression system.
与一般过氧化物酶不同，据报道平菇锰过氧化物酶2（Pleurotus ostreatus MnP2）具有直接氧化高分子量化合物的独特特性，如聚R - 478和核糖核酸酶A（RNase A）。为阐明MnP2氧化聚合底物的机制，利用同源基因表达系统产生了一系列突变酶，并对其反应活性进行了表征。用丙氨酸替代暴露的色氨酸（W170A）的突变酶对藜芦醇（VA）、聚R - 478和RNase A的氧化活性急剧丧失，而对Mn(2+)和H(2)O(2)的动力学性质基本未变。这些结果表明，除了VA外，高分子量底物在W170处也被MnP2直接氧化。此外，在突变体Q266F和V166/168L中，W170周围的氨基酸取代仅导致高分子量底物的活性降低。这些结果，连同突变体的三维建模，表明这些突变对聚合底物进入W170造成了空间位阻。另一个突变体R263N含有一个新产生的N糖基化位点，在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分析中显示出更高的分子量。有趣的是，R263N突变体对VA和高分子量底物的反应活性增加。本文讨论了该突变体中额外的碳水化合物修饰的存在及其催化特性。这是首次使用同源基因表达系统对真菌过氧化物酶氧化高分子量底物的直接机制进行研究。
