title: Detection of a homotetrameric structure and protein-protein interactions of Paracoccidioides brasiliensis formamidase lead to new functional insights.
abstract: Paracoccidioides brasiliensis causes paracoccidioidomycosis, a systemic mycosis in Latin America. Formamidases hydrolyze formamide, putatively plays a role in fungal nitrogen metabolism. An abundant 45-kDa protein was identified as the P. brasiliensis formamidase. In this study, recombinant formamidase was overexpressed in bacteria and a polyclonal antibody to this protein was produced. We identified a 180-kDa protein species reactive to the antibody produced in mice against the P. brasiliensis recombinant purified formamidase of 45 kDa. The 180-kDa purified protein yielded a heat-denatured species of 45 kDa. Both protein species of 180 and 45 kDa were identified as formamidase by peptide mass fingerprinting using MS. The identical mass spectra generated by the 180 and the 45-kDa protein species indicated that the fungal formamidase is most likely homotetrameric in its native conformation. Furthermore, the purified formamidase migrated as a protein of 191 kDa in native polyacrylamide gel electrophoresis, thus revealing that the enzyme forms a homotetrameric structure in its native state. This enzyme is present in the fungus cytoplasm and the cell wall. Use of a yeast two-hybrid system revealed cell wall membrane proteins, in addition to cytosolic proteins interacting with formamidase. These data provide new insights into formamidase structure as well as potential roles for formamidase and its interaction partners in nitrogen metabolism.
authors.affiliation: Laboratório de Biologia Molecular, Instituto de Ciências Biológicas II, Universidade Federal de Goiás, Goiânia, Goiás, Brazil.
authors.forename: Clayton Luiz
authors.initials: CL
authors.lastname: Borges
authors.forename: Juliana Alves
authors.initials: JA
authors.lastname: Parente
authors.forename: Mônica Santiago
authors.initials: MS
authors.lastname: Barbosa
authors.forename: Jaime Martins
authors.initials: JM
authors.lastname: Santa
authors.forename: Sonia Nair
authors.initials: SN
authors.lastname: Báo
authors.forename: Marcelo Valle
authors.initials: MV
authors.lastname: de Sousa
authors.forename: Célia Maria
authors.initials: CM
authors.lastname: de Almeida Soares
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry / Q000378:metabolism*; D015153:Blotting, Western; D002473:Cell Wall / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry; D016253:Microscopy, Immunoelectron; D008970:Molecular Weight; D010228:Paracoccidioides / Q000737:chemistry / Q000201:enzymology*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D020798:Two-Hybrid System Techniques
chemical_list: D000581:Amidohydrolases; C101846:formamidase