Detection of a homotetrameric structure and protein-protein interactions of Paracoccidioides brasiliensis formamidase lead to new functional insights.
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.
Borges
Clayton Luiz
CL
Laboratório de Biologia Molecular, Instituto de Ciências Biológicas II, Universidade Federal de Goiás, Goiânia, Goiás, Brazil.
Parente
Juliana Alves
JA
Barbosa
Mônica Santiago
MS
Santa
Jaime Martins
JM
Báo
Sonia Nair
SN
de Sousa
Marcelo Valle
MV
de Almeida Soares
Célia Maria
CM
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
D000581:Amidohydrolases; C101846:formamidase