RESUMEN
Fumarate reductase is an enzyme involved in maintaining redox balance through regeneration of reduced cofactors during oxygen deficiency conditions. This work reports the identification and characterization of the gene and its promoter and terminator elements that encodes cytosolic fumarate reductase enzyme in the nonconventional yeast, Arxula adeninivorans. The gene harbours an ORF of 1446 bp, encoding a 482-amino acid protein. The deduced amino acid sequence is similar to those of fumarate reductases from other yeast and fungi, such as the two fumarate reductases of Saccharomyces cerevisiae, Frd1p (44%) and Osm1p (41%). This enzyme is located in the cytosol and has a pH optimum of ca. 7.5 and a Michaelis constant (K(M)) of 2.9 mM with fumarate as the substrate. Expression of AFRD1 is regulated by the cultivation conditions. A shift from NaCl-free to NaCl-supplemented media and aerobic to hypoxic growth conditions leads to reduced AFRD1 transcription levels, but not to alteration in the concentration of Afrd1p. The functional analyses of Afrd1p were performed in A. adeninivorans and S. cerevisiae disruption mutants. The A. adeninivorans fumarate reductase is capable of functional complementation of the missing S. cerevisiae genes during anoxia; however, it is not involved in yeast growth under osmotic stress.
Asunto(s)
Proteínas Fúngicas/metabolismo , Saccharomycetales/enzimología , Saccharomycetales/genética , Succinato Deshidrogenasa/metabolismo , Secuencia de Aminoácidos , Hipoxia de la Célula , Clonación Molecular , Medios de Cultivo , ADN de Hongos/genética , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Mutación , Presión Osmótica , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomycetales/crecimiento & desarrollo , Análisis de Secuencia de ADN , Succinato Deshidrogenasa/genéticaRESUMEN
Fumarate reductase is a protein involved in the maintenance of redox balance during oxygen deficiency. This enzyme irreversibly catalyzes the reduction of fumarate to succinate and requires flavin cofactors as electron donors. Two examples are the soluble mitochondrial and the cytosolic fumarate reductases of Saccharomyces cerevisiae encoded by the OSM1 and FRDS1 genes, respectively. This work reports the identification and characterization of the gene encoding cytosolic fumarate reductase enzyme in the arbuscular mycorrhizal fungus, Glomus intraradices and the establishment of its physiological role. Using a yeast expression system, we demonstrate that G. intraradices GiFRD encodes a protein that has fumarate reductase activity which can functionally substitute for the S. cerevisiae fumarate reductases. Additionally, we showed that GiFRD transformants are not affected by presence of salt in medium, indicating that the presence of this gene has no effect on yeast behavior under osmotic stress. The fact that GiFRD expression and enzymatic activity was present only in asymbiotic stage confirmed existence of at least one anaerobic metabolic pathway in this phase of fungus life cycle. This suggests that the AMF behave as facultative anaerobes in the asymbiotic stage.
