title: Analysis of a polygalacturonase gene of Ustilago maydis and characterization of the encoded enzyme.
abstract: 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.
authors.affiliation: Departamento de Biología, División de Ciencias Naturales y Exactas, Campus Guanajuato, Universidad de Guanajuato. Noria Alta s/n, Guanajuato, Gto., México.
authors.forename: José P
authors.initials: JP
authors.lastname: Castruita-Domínguez
authors.forename: Sandra E
authors.initials: SE
authors.lastname: González-Hernández
authors.forename: Julio
authors.initials: J
authors.lastname: Polaina
authors.forename: Lérida L
authors.initials: LL
authors.lastname: Flores-Villavicencio
authors.forename: Aurelio
authors.initials: A
authors.lastname: Alvarez-Vargas
authors.forename: Alberto
authors.initials: A
authors.lastname: Flores-Martínez
authors.forename: Patricia
authors.initials: P
authors.lastname: Ponce-Noyola
authors.forename: Carlos A
authors.initials: CA
authors.lastname: Leal-Morales
mesh_terms: D002244:Carbon / Q000378:metabolism; D003001:Cloning, Molecular; D003470:Culture Media / Q000737:chemistry; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D020869:Gene Expression Profiling; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D010935:Plant Diseases / Q000382:microbiology; D011096:Polygalacturonase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013696:Temperature; D014588:Ustilago / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D003313:Zea mays / Q000382:microbiology
chemical_list: D003470:Culture Media; D004271:DNA, Fungal; D011994:Recombinant Proteins; D002244:Carbon; D011096:Polygalacturonase
keywords: Pgu1 gene expression; Plant tissue; Polygalacturonic acid