Structural and Kinetic Analysis of Schwanniomyces occidentalis Invertase Reveals a New Oligomerization Pattern and the Role of Its Supplementary Domain in Substrate Binding

Abstract

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...

Structural Analysis of β-Fructofuranosidase from Xanthophyllomyces dendrorhous Reveals Unique Features and the Crucial Role of N-Glycosylation in Oligomerization and Activity

“…This construct was used as a template to obtain all the mutants generated in this work. Site-directed mutagenesis was carried out using specific primers, including substitutions responsible for mutations N58S, D80A, N107S, E303A, E334S/E334N/E334V/E334Q, Q341N, N342S, H343A/H343T, N471S, and Y659STOP, and following the method described previously (21). DNA sequencing was used to verify that only the desired mutations were present in all the obtained constructs.…”

“…In the last decade, GH32 enzymes from bacteria (13,14), fungi (15)(16)(17), and plants (18 -20) have been the subject of many structural studies that showed that all are monomeric enzymes. Surprisingly, studies on Schwanniomyces occidentalis fructofuranosidase (SoFfase) (21) and Saccharomyces cerevisiae invertase (ScINV) (22) showed that these enzymes from yeast are unique in that they form dimers mediated by their ␤-sandwich domain, which in the case of ScINV associate into higher oligomers. The structural studies have delineated the main structural determinants of activity, the loops and turns connecting the different elements of the ␤-propeller domain, in the surroundings of the active site, defining the specificity unique to each enzyme (21,23).…”

“…Surprisingly, studies on Schwanniomyces occidentalis fructofuranosidase (SoFfase) (21) and Saccharomyces cerevisiae invertase (ScINV) (22) showed that these enzymes from yeast are unique in that they form dimers mediated by their ␤-sandwich domain, which in the case of ScINV associate into higher oligomers. The structural studies have delineated the main structural determinants of activity, the loops and turns connecting the different elements of the ␤-propeller domain, in the surroundings of the active site, defining the specificity unique to each enzyme (21,23). However, and remarkably, oligomerization of the yeast enzymes regulates their functionality.…”

Semirational Directed Evolution of Loop Regions in Aspergillus japonicus β-Fructofuranosidase for Improved Fructooligosaccharide Production

“…Higher 1-kestose levels for the parent highlighted the difference in activities, as this is the initial FOS species produced from sucrose. It in turn serves as the substrate for the formation of nystose and GF 4 . The fructose data for both enzymes were similar, which indicated that the hydrolytic activity was unchanged in the variant.…”

“…Also known as invertases (EC 3.2.1.26), they hydrolyze sucrose to produce invert sugar, an equimolar mixture of dextrorotatory D-glucose and levorotatory D-fructose (2). Crystal structures reveal that these enzymes display the bimodular arrangement of an N-terminal catalytic domain containing a fivebladed ␤-propeller fold linked to a C-terminal ␤-sandwich domain (3)(4)(5)(6). Multiple sequence alignments (MSAs) identified a highly conserved aspartate close to the N terminus that serves as the catalytic nucleophile and a glutamate residue that acts as a general acid/base catalyst (7).…”