beta-Fructofuranosidases are powerful tools in industrial biotechnology. We have characterized an extracellular beta-fructofuranosidase from the yeast Schwanniomyces occidentalis. The enzyme shows broad substrate specificity, hydrolyzing sucrose, 1-kestose, nystose and raffinose, with different catalytic efficiencies (k(cat)/K(m)). Although the main reaction catalysed by this enzyme is sucrose hydrolysis, it also produces two fructooligosaccharides (FOS) by transfructosylation. A combination of (1)H, (13)C and 2D-NMR techniques shows that the major product is the prebiotic trisaccharide 6-kestose. The 6-kestose yield obtained with this beta-fructofuranosidase is, to our concern, higher than those reported with other 6-kestose-producing enzymes, both at the kinetic maximum (76gl(-1)) and at reaction equilibrium (44gl(-1)). The total FOS production in the kinetic maximum was 101gl(-1), which corresponded to 16.4% (w/w) referred to the total carbohydrates in the reaction mixture.
Grx5 defines a family of yeast monothiol glutaredoxins that also includes Grx3 and Grx4. All three proteins display significant sequence homology with proteins found from bacteria to humans. Grx5 is involved in iron/ sulfur cluster assembly at the mitochondria, but the function of Grx3 and Grx4 is unknown. Three-dimensional modeling based on known dithiol glutaredoxin structures predicted a thioredoxin fold structure for Grx5. Positionally conserved amino acids in this glutaredoxin family were replaced in Grx5, and the effect on the biological function of the protein has been tested. For all changes studied, there was a correlation between the effects on several different phenotypes: sensitivity to oxidants, constitutive protein oxidation, ability for respiratory growth, auxotrophy for a number of amino acids, and iron accumulation. Cys 60 and Gly 61 are essential for Grx5 function, whereas other single or double substitutions in the same region had no phenotypic effects. Gly 115 and Gly 116 could be important for the formation of a glutathione cleft on the Grx5 surface, in contrast to adjacent Cys 117 . Substitution of Phe 50 alters the -sheet in the thioredoxin fold structure and inhibits Grx5 function. None of the substitutions tested affect the structure at a significant enough level to reduce protein stability.Glutaredoxins are thiol oxidireductases that catalyze redox reactions involving reduced glutathione as a hydrogen donor for the reduction of protein disulfides (dithiol mechanism of action) or glutathione-protein-mixed disulfides (monothiol mechanism of action) (see Refs. 1 and 2 for review). Previously described glutaredoxins are small proteins (about 10 kDa) with a conserved active site that includes two cysteine residues (Cys-Pro-Tyr-Cys). Site-directed mutagenesis (3-5) has demonstrated that both cysteine residues in the active site are required for the dithiol reaction. In contrast, the amino-terminal cysteine is sufficient to catalyze the deglutathionylation of the reduced glutathione-mixed disulfides that are formed under oxidative stress conditions (5).Three-dimensional structures of oxidized and reduced forms of viral, bacterial, and mammalian glutaredoxins and also of reduced glutathione-glutaredoxin complexes have been identified using x-ray crystallography (6, 7) or nuclear magnetic resonance spectroscopy (8 -14). These studies have revealed which residues, apart from those at the active site, are important for stable interactions between glutathione and the glutaredoxin molecule (10,13,14). Dithiol glutaredoxins are members of the thioredoxin superfamily (15, 16) along with at least five other classes of proteins that interact with cysteine-containing substrates (thioredoxins, DbsA, protein disulfide isomerases, glutathione S-transferases, and glutathione peroxidases). This superfamily shares a structural motif (called the thioredoxin fold or ␣␣ fold) formed by a four or fivestranded -sheet (with parallel and antiparallel strands) surrounded by three or more ␣-helices distributed on ei...
Fungal laccases are remarkable green catalysts that have a broad substrate specificity and many potential applications in bioremediation, lignocellulose processing, organic synthesis, and more. However, most of these transformations must be carried out at high concentrations of organic cosolvents in which laccases undergo unfolding, thereby losing their activity. We have tailored a thermostable laccase that tolerates high concentrations of cosolvents, the genetic product of five rounds of directed evolution expressed in Saccharomyces cerevisiae. This evolved laccase--R2 variant--was capable of resisting a wide array of cosolvents at concentrations as high as 50% (v/v). Intrinsic laccase features such as the redox potential and the geometry of catalytic copper varied slightly during the course of the molecular evolution. Some mutations at the protein surface stabilized the laccase by allowing additional electrostatic and hydrogen bonding to occur.
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