title: Identification, characterization and regulation studies of the aconitase of Paracoccidioides brasiliensis.
abstract: A protein species preferentially expressed in yeast cells with a molecular mass of 80 kDa and isoeletric point (pI) of 7.79 was isolated from the proteome of Paracoccidioides brasiliensis and characterized as an aconitase (ACO) (E.C. 4.2.1.3). ACO is an enzyme that catalyzes the isomerization of citrate to isocitrate in both the Krebs cycle and the glyoxylate cycle. We report the cloning and characterization of the cDNA encoding the ACO of P. brasiliensis (PbACO). The cDNA showed a 2361 bp open reading frame (ORF) and encoded a predicted protein with 787 amino acids. Polyclonal antibodies against the purified recombinant PbACO was obtained in order to analyze the subcellular localization of the molecule in P. brasiliensis. The protein is present in the extracellular fluid, cell wall enriched fraction, mitochondria, cytosol and peroxisomes of yeast cells as demonstrated by western blot and immunocytochemistry analysis. The expression analysis of the Pbaco gene was performed by quantitative real-time RT-PCR and results demonstrated an increased expression in yeast cells compared to mycelia. Real-time RT-PCR assays was also used to evaluate the Pbaco expression when the fungus grows on media with acetate and ethanol as sole carbon sources and in different iron levels. The results demonstrated that Pbaco transcript is over expressed in acetate and ethanol as sole carbon sources and in high-iron conditions.
authors.affiliation: Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia, Goiás, Brazil.
authors.forename: Wesley de A
authors.initials: Wde A
authors.lastname: Brito
authors.forename: Tereza Cristina V
authors.initials: TC
authors.lastname: Rezende
authors.forename: Ana Flávia
authors.initials: AF
authors.lastname: Parente
authors.forename: Carlos André O
authors.initials: CA
authors.lastname: Ricart
authors.forename: Marcelo V de
authors.initials: MV
authors.lastname: Sousa
authors.forename: Sônia N
authors.initials: SN
authors.lastname: Báo
authors.forename: Célia Maria de A
authors.initials: CM
authors.lastname: Soares
mesh_terms: D000154:Aconitate Hydratase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D007501:Iron / Q000378:metabolism; D008969:Molecular Sequence Data; D025282:Mycelium / Q000737:chemistry / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D010228:Paracoccidioides / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D021381:Protein Transport
chemical_list: D005656:Fungal Proteins; D007501:Iron; D000154:Aconitate Hydratase