The Three-dimensional Structure of Invertase (β-Fructosidase) from Thermotoga maritima Reveals a Bimodular Arrangement and an Evolutionary Relationship between Retaining and Inverting Glycosidases

Background:The catalytic mechanism and substrate recognition required for cleavage of the ␣-linkage in agarose are unclear.Results: Structural analysis of a family 117 glycoside hydrolase details substrate recognition and supports an inverting mechanism. Conclusion: GH117 enzymes use substrate distortion and an unusual general acid for catalysis. Significance: Microbes may utilize alternate strategies to catalyze the degradation of polysaccharides with unique structural characteristics.

“…2C). In both of these inverting enzymes, the general base has been predicted as Asp 38 in Arb43A and Asp 15 in Xyn3B. In BpGH117, the residue Asp 90 aligns well with these two residues.…”

Section: Bpgh117mentioning

confidence: 94%

Section: Bpgh117mentioning

confidence: 99%

Analysis of Keystone Enzyme in Agar Hydrolysis Provides Insight into the Degradation (of a Polysaccharide from) Red Seaweeds

Hehemann

Smyth

Yadav

et al. 2012

“…cazy.org/CAZY/index.html) place these plant and microbial hydrolytic enzymes into the GH32 family (O-glycoside hydrolases). Structural analyses of the GH32 enzymes invertase (8,9), exo-inulinase (10), and plant exohydrolase (11) have revealed substantial structural similarities; these enzymes consist of fivebladed ␤-propeller domains connected to ␤-sandwich domains. In addition, active sites for the proposed hydrolytic activity have been identified; they include three acidic amino acid residues.…”

mentioning

confidence: 99%

Structural and Functional Insights into Intramolecular Fructosyl Transfer by Inulin Fructotransferase

Jung

Hong²,

Lee

et al. 2007

Inulin fructotransferase (IFTase), a member of glycoside hydrolase family 91, catalyzes depolymerization of ␤-2,1-fructans inulin by successively removing the terminal difructosaccharide units as cyclic anhydrides via intramolecular fructosyl transfer. The crystal structures of IFTase and its substratebound complex reveal that IFTase is a trimeric enzyme, and each monomer folds into a right-handed parallel ␤-helix. Despite variation in the number and conformation of its ␤-strands, the IFTase ␤-helix has a structure that is largely reminiscent of other ␤-helix structures but is unprecedented in that trimerization is a prerequisite for catalytic activity, and the active site is located at the monomer-monomer interface. Results from crystallographic studies and site-directed mutagenesis provide a structural basis for the exolytic-type activity of IFTase and a functional resemblance to inverting-type glycosyltransferases.Fructans are polysaccharides composed of linear and branched polymers of fructose linked to sucrose through glycosidic bonds of various linkage types. They have been conceived as one of the principal stored forms of energy in 15% of higher plants, as well as in a wide range of bacteria and fungi (1). Several plant fructosyltransferases, each with a distinct substrate and glycosidic bond linkage-type specificity, have been suggested to be involved in the sequential enzymatic steps that produce fructans such as ␤-2,6-linked levan and ␤-2,1-linked inulin. In this process, fructose is first linked to vacuolar sucrose, and then fructosyl units are successively added to the resulting trisaccharide (1, 2). Not only do plant fructans play a major role as storage carbohydrates, but they are also implicated in additional physiological functions in plants, such as drought and cold tolerance (1). By contrast, in bacteria, the multifunctional enzymes levansucrase (3) and inulosucrase (4) catalyze fructan biosynthesis, producing inulin and levan, the predominant bacterial fructans, respectively. Details of the levan biosynthetic mechanism in bacteria were recently revealed by structural studies of levansucrase (3, 5).Fructan-degrading enzymes that function primarily in the mobilization of stored fructans in plants and microbes have also been characterized. Just recently, the plant fructan hydrolases (EC 3.2.1) were found to catalyze the hydrolysis of levan and inulin via an exclusively exolytic mechanism that releases successive terminal fructose units (6). The presence of these fructan exohydrolases, even in non-fructan-containing plants, suggests an additional, defensive role for these enzymes against pathogenic bacteria (2).In bacteria, two distinctly different classes of enzymes perform fructan degradation. One of these classes includes two hydrolases, levanase (EC 3.2.1.65) and inulinase (EC 3.2.1.7), which exhibit both endo-and exotype hydrolytic activities. Classifications based on sequence similarity (7) (CAZy; www. cazy.org/CAZY/index.html) place these plant and microbial hydrolytic enzymes into the GH32...

“…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 into the GHJ clan together with the GH68 (inulosucrase family).…”

mentioning

confidence: 99%

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

Álvaro-Benito

Polo

González

et al. 2010

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...