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

Abstract

Xanthophyllomyces dendrorhous ␤-fructofuranosidase (XdINV) is a highly glycosylated dimeric enzyme that hydrolyzes sucrose and releases fructose from various fructooligosaccharides (FOS) and fructans. It also catalyzes the synthesis of FOS, prebiotics that stimulate the growth of beneficial bacteria in human gut. In contrast to most fructosylating enzymes, XdINV produces neo-FOS, which makes it an interesting biotechnology target. We present here its three-dimensional structure, which shows the expected bimodular arrangement and also a long extension of its C terminus that together with an N-linked glycan mediate the formation of an unusual dimer. The two active sites of the dimer are connected by a long crevice, which might indicate its potential ability to accommodate branched fructans. This arrangement could be representative of a group of GH32 yeast enzymes having the traits observed in XdINV. The inactive D80A mutant was used to obtain complexes with relevant substrates and products, with their crystals structures showing at least four binding subsites at each active site. Moreover, two different positions are observed from subsite ؉2 depending on the substrate, and thus, a flexible loop (Glu-334 -His-343) is essential in binding sucrose and ␤(2-1)-linked oligosaccharides. Conversely, ␤(2-6) and neo-type substrates are accommodated mainly by stacking to Trp-105, explaining the production of neokestose and the efficient fructosylating activity of XdINV on ␣-glucosides. The role of relevant residues has been investigated by mutagenesis and kinetics measurements, and a model for the transfructosylating reaction has been proposed. The plasticity of its active site makes XdINV a valuable and flexible biocatalyst to produce novel bioconjugates.

Fructosylation of Hydroxytyrosol by the β‐Fructofuranosidase from Xanthophyllomyces dendrorhous: Insights into the Molecular Basis of the Enzyme Specificity

“…A detailed view of the precise ligand binding in both complexes is shown in Figure . In the ternary complex (Figure a), HT1 is bound by stacking its aromatic ring against Trp105; interestingly, this hydrophobic contact is the driving force directing sucrose binding in the catalytic pocket as to make neokestose, the main transfructosylation product of Xd–INV . This feature is in agreement with a preferred binding of hydroxytyrosol at HT1 position with the concomitant production of the observed major derivative.…”

“…In the ternary intermediate complex (Figure a), β‐fructose occupies subsite −1 in a position conserved with that previously described . Interestingly, there are two hydroxytyrosol molecules (HT1 and HT2 in the figure) that are located in positions appropriate to act as potential acceptors of β‐fructose, as both substrates present OH groups in the proximity of the fructose C2 to make the glycosidic linkage.…”

“…Additionally, this enzyme disclosed the ability to fructosylate other carbohydrates containing a glucose moiety such as maltose and maltotriose . Moreover, the presence of a HEPES molecule in the crystals, mimicking the location of an acceptor for the fructose in a putative intermediate complex, suggested the possibility of testing other non‐sugar compounds as acceptor substrates.…”

“…A remarkable finding of our crystallographic analysis was the presence of a HEPES molecule in the crystals, coming from the buffer solution . This HEPES molecule was located in the active site next to a fructose unit, in an appropriate position that showed its potential as an acceptor.…”

“…Xd–INV is a dimeric protein with a molecular weight of 320–380 kDa, 60–70 % of which corresponds to N‐linked carbohydrates. We reported the crystal structure of the native enzyme, which revealed that the glycan chains attached to two specific glycosylation sites are crucial for dimerization and activity . Furthermore, the structural analysis of complexes of the inactive mutant (pXd‐INV‐D80A) with various short fructooligosaccharides acting as potential substrates or products uncovered the molecular basis of its broad substrate specificity, and pointed to the key role of some particular residues in the substrate binding.…”