Crystal Structures of Aspergillus japonicus Fructosyltransferase Complex with Donor/Acceptor Substrates Reveal Complete Subsites in the Active Site for Catalysis

Abstract

Fructosyltransferases catalyze the transfer of a fructose unit from one sucrose/fructan to another and are engaged in the production of fructooligosaccharide/fructan. The enzymes belong to the glycoside hydrolase family 32 (GH32) with a retaining catalytic mechanism. Here we describe the crystal structures of recombinant fructosyltransferase (AjFT) from Aspergillus japonicus CB05 and its mutant D191A complexes with various donor/acceptor substrates, including sucrose, 1-kestose, nystose, and raffinose. This is the first structure of fructosyltransferase of the GH32 with a high transfructosylation activity. The structure of AjFT comprises two domains with an N-terminal catalytic domain containing a five-blade ␤-propeller fold linked to a C-terminal ␤-sandwich domain. Structures of various mutant AjFT-substrate complexes reveal complete four substrate-binding subsites (؊1 to ؉3) in the catalytic pocket with shapes and characters distinct from those of clan GH-J enzymes. Residues Asp-60, Asp-191, and Glu-292 that are proposed for nucleophile, transition-state stabilizer, and general acid/base catalyst, respectively, govern the binding of the terminal fructose at the ؊1 subsite and the catalytic reaction. Mutants D60A, D191A, and E292A completely lost their activities. Residues Ile-143, Arg-190, Glu-292, Glu-318, and His-332 combine the hydrophobic Phe-118 and Tyr-369 to define the ؉1 subsite for its preference of fructosyl and glucosyl moieties. Ile-143 and Gln-327 define the ؉2 subsite for raffinose, whereas Tyr-404 and Glu-405 define the ؉2 and ؉3 subsites for inulin-type substrates with higher structural flexibilities. Structural geometries of 1-kestose, nystose and raffinose are different from previous data. All results shed light on the catalytic mechanism and substrate recognition of AjFT and other clan GH-J fructosyltransferases.

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

“…In contrast, the substrate is loosely bound at subsite ϩ2 or dangling into the solvent. In agreement with this observation, short substrates such as 1-kestose or raffinose are accommodated in essentially the same conformation in all the formerly described complexes (13,16,18,19). In contrast, XdINV presents a deep cavity showing multiple binding sites, a feature that has been previously described for two other fungal enzymes, the AjFT and the SoFfase.…”

“…The reported complexes of these enzymes with long substrates reveal that the active sites diverge from subsite ϩ2, and thus, nystose bound at the AjFT catalytic pocket locates its terminal glucose at a position that is occupied by Trp-105 in XdINV (see Fig. 7A) (16). A very short T I loop and the absence of the C-terminal segment make a very different active site pocket in AjFT, which is remarkable taking into account that this enzyme is phylogenetically close to XdINV.…”

“…A distinctive trait of GH32 enzymes is the presence of an additional ␤-sandwich structure fused at the C terminus of the catalytic domain. 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.…”

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

“…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).…”

“…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). It is well known that some ␤-fructofuranosidases possess fructosyltransferase activity whereby the sugar moiety is transferred from the enzyme-fructosyl intermediate to a substrate other than water (6,8). This reaction forms the basis of fructooligosaccharide (FOS) synthesis from sucrose.…”