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Fructooligosaccharide is a mixture of mostly the trisaccharide 1-kestose (GF2), tetrasaccharide nystose (GF3), and fructosyl nystose (GF4). Enzymes that hydrolyze GF3 may be useful for preparing GF2 from the fructooligosaccharide mixture. A β-fructofuranosidase belonging to glycoside hydrolase family 32 (GH32) from the honeybee gut bacterium Frischella perrara (FperFFase) was expressed in Escherichia coli and purified. The time course of the hydrolysis of 60 mM sucrose, GF2, and GF3 by FperFFase was analyzed, showing that the hydrolytic activity of FperFFase for trisaccharide GF2 was lower than those for disaccharide sucrose and tetrasaccharide GF3. The crystal structure of FperFFase and its structure in complex with fructose were determined. FperFFase was found to be structurally homologous to bifidobacterial β-fructofuranosidases even though bifidobacterial enzymes preferably hydrolyze GF2 and the amino acid residues interacting with fructose at subsite − 1 are mostly conserved between them. A proline residue was inserted between Asp298 and Ser299 using site-directed mutagenesis, and the activity of the variant 298P299 was measured. The ratio of activities for 60 mM GF2/GF3 by wild-type FperFFase was 35.5%, while that of 298P299 was 23.6%, indicating that the structure of the loop comprising Trp297-Asp298-Ser299 correlated with the substrate preference of FperFFase. The crystal structure also shows that a loop consisting of residues 117–127 is likely to contribute to the substrate binding of FperFFase. The results obtained herein suggest that FperFFase is potentially useful for the manufacture of GF2. Key points: • Frischella β-fructofuranosidase hydrolyzed nystose more efficiently than 1-kestose. • Trp297-Asp298-Ser299 was shown to be correlated with the substrate preference. • Loop consisting of residues 117–127 appears to contribute to the substrate binding. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus exfoliative toxins (ETs) are serine proteases responsible for staphylococcal scalded skin syndrome. Four ETs, ETA, ETB, ETD, and ETE, have been identified, all of which cleave desmoglein-1. This study presents the crystal structure of ETD at 1.75 Å resolution. The protein exhibits a structure composed of two β-barrels and two α-helices as described in previous studies of ETs. A predicted model of ETD in complex with Ile380–Glu381–Gly382–Pro383 (IEGP), a segment of human desmoglein-1 (hDsg1), was constructed. Glu381 of hDsg1 was predicted to interact with as many as six amino acid residues in ETD, whereas two amino acid residues in ETD primarily constituted subsite S1′, and a space near subsite S1′ was noted. It is likely that polypeptide chains located near the IEGP segment in the predicted structure of hDsg1 bind to this space. The structure of loop D, which was predicted to participate in subsite S2′, in ETD was markedly different from those in other ETs. • A crystal structure of exfoliative toxin D (ETD) was determined at 1.75 Å resolution. • A model of ETD in complex with part of human desmoglein-1 (hDsg1) was constructed. • Glu381 in hDsg1 interacted with six amino acid residues in ETD. • A space near subsite S1′ potentially binds to other parts of ETD. • The ETD loop D structure was different from those in the other exfoliative toxins. [ABSTRACT FROM AUTHOR]