Schwanniomyces occidentalis invertase is an extracellular enzyme that hydrolizes sucrose and releases ␤-fructose from various oligosaccharides and essential storage fructan polymers such as inulin. We report here the three-dimensional structure of Sw. occidentalis invertase at 2.9 Å resolution and its complex with fructose at 1.9 Å resolution. The monomer presents a bimodular arrangement common to other GH32 enzymes, with an N-terminal 5-fold ␤-propeller catalytic domain and a C-terminal ␤-sandwich domain for which the function has been unknown until now. However, the dimeric nature of Sw. occidentalis invertase reveals a unique active site cleft shaped by both subunits that may be representative of other yeast enzymes reported to be multimeric. Binding of the tetrasaccharide nystose and the polymer inulin was explored by docking analysis, which suggested that medium size and long substrates are recognized by residues from both subunits. The identified residues were mutated, and the enzymatic activity of the mutants against sucrose, nystose, and inulin were investigated by kinetic analysis. The replacements that showed the largest effect on catalytic efficiency were Q228V, a residue putatively involved in nystose and inulin binding, and S281I, involved in a polar link at the dimer interface. Moreover, a significant decrease in catalytic efficiency against inulin was observed in the mutants Q435A and Y462A, both located in the ␤-sandwich domain of the second monomer. This highlights the essential function that oligomerization plays in substrate specificity and assigns, for the first time, a direct catalytic role to the supplementary domain of a GH32 enzyme.Fructans, the fructose-rich polymers derived biosynthetically from sucrose, are important storage oligosaccharides and polysaccharides in many bacteria and fungi and numerous plant species. Furthermore, sucrose is one of the most widespread disaccharides in nature and is especially ubiquitous in higher plants as the first free sugar resulting from photosynthesis. It is the major transport compound to bring energy and carbon skeletons from source to sink tissues. Carbohydrate partitioning and sugar sensing are intimately connected to sucrose metabolism; these processes are vital throughout plant development. Therefore, the enzymes involved in fructans and sucrose processing are essential to plant cell metabolism.The enzymes that hydrolyze sucrose are referred to collectively as invertases or ␤-fructofuranosidases (EC 3.2.1.26) and catalyze the release of ␤-fructose from the nonreducing end of various ␤-D-fructofuranoside substrates (Fig. 1). The cleavage of the ␤-glycosidic bond is carried out by a double displacement catalytic mechanism that retains the configuration of the fructose anomeric carbon, two conserved residues, an aspartic and a glutamic acid, being the nucleophile and the general acid-base catalyst, respectively. On the basis of the amino acid sequences (1) they are classified into family 32 of the glycosylhydrolases (GH32), 3 which are included in...

The soil acid-base buffering capacity and the biological availability, mobilization, and transport of macro- and micronutrients, toxic metal ions, and xenobiotic organic cations in soil are strongly influenced by the acid-base properties of humic substances, of which humic and fulvic acids are the major fractions. For these reasons, the proton binding behavior of the humic acid-like (HA) and fulvic acid-like (FA) fractions contained in a compost are believed to be instrumental in its successful performance in soil. In this work, the acid-base properties of the HAs and FAs isolated from a mixture of the sludge residue obtained from olive oil mill wastewater (OMW) evaporated in an open-air pond and tree cuttings (TC) at different stages of composting were investigated by a current potentiometric titration method and the nonideal competitive adsorption (NICA)-Donnan model. The NICA-Donnan model provided an excellent description of the acid-base titration data, and pointed out substantial differences in site density and proton-binding affinity between the HAs and FAs examined. With respect to FAs, HAs were characterized by a smaller content of carboxylic- and phenolic-type groups and their larger affinities for proton binding. Further, HAs featured a greater heterogeneity in carboxylic-type groups than FAs. The composting process increased the content and decreased the proton affinity of carboxylic- and phenolic-type groups of HAs and FAs, and increased the heterogeneity of phenolic-type groups of HAs. As a whole, these effects indicated that the composting process could produce HA and FA fractions with greater cation binding capacities. These results suggest that composting of organic materials improves their agronomic and environmental value by increasing their potential to retain and exchange macro- and micronutrients, and to reduce the bioavailability of organic and inorganic pollutants.

Schwanniomyces occidentalis invertase is an extracellular enzyme that releases beta-fructose from the nonreducing termini of various beta-d-fructofuranoside substrates. Its ability to produce 6-kestose by transglycosylation makes this enzyme an interesting research target for applications in industrial biotechnology. The enzyme has been expressed in Saccharomyces cerevisiae. Recombinant and wildtype forms, which showed different glycosylation patterns, were crystallized by vapour-diffusion methods. Although crystallization trials were conducted on both forms of the protein, crystals suitable for X-ray crystallographic analyses were only obtained from the wild-type enzyme. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 105.78, b = 119.49, c = 137.68 angstrom. A diffraction data set was collected using a synchrotron source. Self-rotation function and sedimentation-velocity experiments suggested that the enzyme was dimeric with twofold symmetry.