The essential role of Glu-185 and Tyr-354 residues in the ferroxidase activity of Saccharomyces cerevisiae Fet3

AbstractThe structural determinants required for ferroxidase activity by the yeast multicopper oxidase Fet3 have been partially clarified by site-directed mutagenesis based on homology modeling. Glu-185 and Tyr-354 were substituted with Ala and Phe, respectively. Fet3 E185A retained ca. 5% residual ferroxidase catalytic efficiency, and almost 40% oxidase efficiency. On the other hand, Fet3 Y354F exhibited 50% residual efficiency as a ferroxidase and more than 70% as an oxidase. These results provide new insights in the mechanism of iron binding and oxidation by Fet3, establishing the essential role of Glu-185 and Tyr-354, and allowing to dissect ferroxidase from non-iron oxidase activity

Growth and Primary Metabolism of Lettuce Seedlings (<i>Lactuca sativa</i> L.) Are Promoted by an Innovative Iron-Based Fenton-Composted Amendment

Information regarding the physiological and molecular plant responses to the treatment with new biofertilizers is limited. In this study, a fast-composting soil amendment obtained from solid waste by means of a Fenton reaction was assessed to evaluate the effects on the growth of Lactuca sativa L. var. longifolia seedlings. Growth rate, root biomass, chlorophyll concentration, and total soluble proteins of seedlings treated with the 2% fast-composting soil amendment showed significant increases in comparison with the control seedlings. Proteomic analysis revealed that the soil amendment induced the up-regulation of proteins belonging to photosynthesis machinery, carbohydrate metabolism, and promoted energy metabolism. Root proteomics indicated that the fast-composting soil amendment strongly induced the organs morphogenesis and development; root cap development, lateral root formation, and post-embryonic root morphogenesis were the main biological processes enriched by the treatment. Overall, our data suggest that the addition of the fast-composting soil amendment formulation to the base soils might ameliorate plant growth by inducing carbohydrate primary metabolism and the differentiation of a robust root system

Purification and characterization of recombinant Caulobacter crescentus Cu,Zn superoxide dismutase

Recombinant Cu,Zn Superoxide Dismutase from Caulobacter crescentus has been expressed in Escherichia coli and characterized. The corresponding recombinant protein has a molecular weight typical of a homodimeric Cu,ZnSODs and an activity comparable to that of other prokaryotic enzymes. The copper active site is characterized by a peculiar axial geometry as evidenced by its electron paramagnetic resonance spectrum, moreover, the copper atom displays a low accessibility toward external chelating agents indicating a lower solvent accessibility when compared to other prokaryotic enzymes. Investigation of the enzyme thermal stability through differential scanning calorimetry indicates the occurrence of two transitions at low and higher temperature that are found to be due to the apo and holo protein, respectively, confirming that the metals have a crucial role in the stabilization of this class of enzymes. © 2005 Elsevier B.V. All rights reserved

Mutational analysis of the iron binding site of Saccharomyces cerevisiae ferroxidase Fet3. An in vivo study

The role of residues predicted to be involved in the binding of iron by the yeast ferroxidase Fet3 has been studied by site-directed mutagenesis. The effect of Fet3 mutations E185A, E185Q, Y354F, D409V and H489D has been investigated in vivo by kinetic analyses of high affinity iron uptake. Our results indicate that Glu-185 is critical for the binding of iron, since substitution of this residue with Ala or Gin strongly affects both growth and the kinetic parameters of high affinity iron uptake, greatly increasing K-m. Mutations Y354F and D409V result in less severe alteration of high affinity iron uptake, while mutant H489D is unable to grow under conditions of iron limitation. (C) 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies