Departamento de Biología, División de Ciencias Naturales y Exactas, Campus Guanajuato, Universidad de Guanajuato. Noria Alta s/n, Guanajuato, Gto., México.
J Basic Microbiol. 2014 May;54(5):340-9. doi: 10.1002/jobm.201200606. Epub 2013 May 20.
Ustilago maydis is a pathogenic fungus that produces the corn smut. It is a biotrophic parasite that depends on living plant tissues for its proliferation and development. Polygalacturonases are secreted by pathogens to solubilize the plant cell-wall and are required for pathogen virulence. In this paper, we report the isolation of a U. maydis polygalacturonase gene (Pgu1) and the functional and structural characterization of the encoded enzyme. The U. maydis Pgu1 gene is expressed when the fungus is grown in liquid culture media containing different carbon sources. In plant tissue, the expression increased as a function of incubation time. Pgu1 gene expression was detected during plant infection around 10 days post-infection with U. maydis FB-D12 strain in combination with teliospore formation. Synthesis and secretion of active recombinant PGU1 were achieved using Pichia pastoris, the purified enzyme had a optimum temperature of 34 °C, optimum pH of 4.5, a Km of 57.84 g/L for polygalacturonic acid, and a Vmax of 28.9 µg/min mg. Structural models of PGU1 based on homologous enzymes yielded a typical right-handed β-helix fold of pectinolytic enzymes classified in the glycosyl hydrolases family 28, and the U. maydis PGU1 is related with endo rather than exo polygalacturonases.
玉米黑粉菌是一种致病真菌，可导致玉米黑粉病。它是一种生物寄生菌，依赖于活的植物组织来增殖和发育。多聚半乳糖醛酸酶由病原体分泌，用于溶解植物细胞壁，对于病原体的毒力是必需的。在本文中，我们报告了玉米黑粉菌多聚半乳糖醛酸酶基因（Pgu1）的分离及其编码酶的功能和结构特征。当真菌在含有不同碳源的液体培养介质中生长时，玉米黑粉菌 Pgu1 基因表达。在植物组织中，随着培养时间的增加，其表达量增加。在玉米黑粉菌 FB-D12 菌株感染植物后约 10 天，与担孢子形成相结合时，检测到 Pgu1 基因表达。使用毕赤酵母实现了活性重组 PGU1 的合成和分泌，纯化后的酶最适温度为 34°C，最适 pH 值为 4.5，对聚半乳糖醛酸的 Km 值为 57.84 g/L，Vmax 值为 28.9 µg/min·mg。基于同源酶的 PGU1 结构模型产生了果胶水解酶家族 28 中分类的典型右手β-螺旋折叠，而玉米黑粉菌 PGU1 与内切多聚半乳糖醛酸酶而不是外切多聚半乳糖醛酸酶有关。
