Crystal structure of a ß-fructofuranosidase with high transfructosylation activity from Aspergillus kawachii.

ß-Fructofuranosidases belonging to glycoside hydrolase family (GH) 32 are enzymes that hydrolyze sucrose. Some GH32 enzymes also catalyze transfructosylation to produce fructooligosaccharides. We found that Aspergillus kawachii IFO 4308 ß-fructofuranosidase (AkFFase) produces fructooligosaccharides, mainly 1-kestose, from sucrose. We determined the crystal structure of AkFFase. AkFFase is composed of an N-terminal small component, a ß-propeller catalytic domain, an α-helical linker, and a C-terminal ß-sandwich, similar to other GH32 enzymes. AkFFase forms a dimer, and the dimerization pattern is different from those of other oligomeric GH32 enzymes. The complex structure of AkFFase with fructose unexpectedly showed that fructose binds both subsites -1 and +1, despite the fact that the catalytic residues were not mutated. Fructose at subsite +1 interacts with Ile146 and Glu296 of AkFFase via direct hydrogen bonds.

A glycoside hydrolase family 31 dextranase with high transglucosylation activity from Flavobacterium johnsoniae.

Glycoside hydrolase family (GH) 31 enzymes exhibit various substrate specificities, although the majority of members are α-glucosidases. Here, we constructed a heterologous expression system of a GH31 enzyme, Fjoh_4430, from Flavobacterium johnsoniae NBRC 14942, using Escherichia coli, and characterized its enzymatic properties. The enzyme hydrolyzed dextran and pullulan to produce isomaltooligosaccharides and isopanose, respectively. When isomaltose was used as a substrate, the enzyme catalyzed disproportionation to form isomaltooligosaccharides. The enzyme also acted, albeit inefficiently, on p-nitrophenyl α-D-glucopyranoside, and p-nitrophenyl α-isomaltoside was the main product of the reaction. In contrast, Fjoh_4430 did not act on trehalose, kojibiose, nigerose, maltose, maltotriose, or soluble starch. The optimal pH and temperature were pH 6.0 and 60 °C, respectively. Our results indicate that Fjoh_4430 is a novel GH31 dextranase with high transglucosylation activity.

Structural insights into polysaccharide recognition by Flavobacterium johnsoniae dextranase, a member of glycoside hydrolase family 31.

Glycoside hydrolase family (GH) 31 contains a large variety of enzymes, but the major members are enzymes that act on relatively small oligosaccharides such as α-glucosidase. Here, we determined the crystal structure of Flavobacterium johnsoniae dextranase (FjDex31A), an enzyme from F. johnsoniae that hydrolyzes a polysaccharide, dextran. FjDex31A is composed of four domains: an N-terminal domain, a catalytic domain, a proximal C-terminal domain, and a distal C-terminal domain, as observed in typical GH31 enzymes. However, the architecture of active site residues in FjDex31A, other than subsite -1, is markedly different from that of other GH31 enzymes. The FjDex31A structure in complex with isomaltotriose shows that Gly273 and Tyr524, both of which interact with an α-glucose residue at subsite -2, as well as Trp376 and Leu308-cisGln309, are especially unique to FjDex31A. Site-directed mutagenesis of Gly273 and Tyr524 resulted in a decrease in the hydrolysis of polysaccharides dextran and pullulan, as well as that of the disaccharide isomaltose. These results suggest that, regardless of the length of sugar chains of the substrates, binding of FjDex31A to the substrates at subsite -2 is likely to be important for its activity. DATABASE: Structural data are available in the Protein Data Bank under the accession numbers 6JR6, 6JR7, and 6JR8.