Glycoside hydrolase family 68 (GH68) enzymes catalyze β-fructosyltransfer from sucrose to another sucrose, the so-called transfructosylation. Although regioselectivity of transfructosylation is divergent in GH68 enzymes, there is insufficient information available on the structural factor(s) involved in the selectivity. Here, we found two GH68 enzymes, β-fructofuranosidase (FF Zm ) and levansucrase (LS Zm ), encoded tandemly in the genome of Zymomonas mobilis , displayed different selectivity: FF Zm catalyzed the β-(2→1)-transfructosylation (1-TF), whereas LS Zm did both of 1-TF and β-(2→6)-transfructosylation (6-TF). We identified His79 FF Zm and Ala343 FF Zm and their corresponding Asn84 LS Zm and Ser345 LS Zm respectively as the structural factors for those regioselectivities. LS Zm with the respective substitution of FF Zm -type His and Ala for its Asn84 LS Zm and Ser345 LS Zm (N84H/S345A-LS Zm ) lost 6-TF and enhanced 1-TF. Conversely, the LS Zm -type replacement of His79 FF Zm and Ala343 FF Zm in FF Zm (H79N/A343S-FF Zm ) almost lost 1-TF and acquired 6-TF. H79N/A343S-FF Zm exhibited the selectivity like LS Zm but did not produce the β-(2→6)-fructoside-linked levan and/or long levanooligosaccharides that LS Zm did. We assumed Phe189 LS Zm to be a responsible residue for the elongation of levan chain in LS Zm and mutated the corresponding Leu187 FF Zm in FF Zm to Phe. An H79N/L187F/A343S-FF Zm produced a higher quantity of long levanooligosaccharides than H79N/A343S-FF Zm (or H79N-FF Zm ), although without levan formation, suggesting that LS Zm has another structural factor for levan production. We also found that FF Zm generated a sucrose analog, β-D-fructofuranosyl α-D-mannopyranoside, by β-fructosyltransfer to d -mannose and regarded His79 FF Zm and Ala343 FF Zm as key residues for this acceptor specificity. In summary, this study provides insight into the structural factors of regioselectivity and acceptor specificity in transfructosylation of GH68 enzymes.

1The recombinant catalytic α-subunit of N-glycan processing glucosidase II from 2 Schizosaccharomyces pombe (SpGIIα) was produced in Escherichia coli. The recombinant 3 SpGIIα exhibited quite low stability, with a reduction in activity to < 40% after 2-days 4 preservation at 4°C, but the presence of 10% (v/v) glycerol prevented this loss of activity. 5 SpGIIα, a member of the glycoside hydrolase family 31 (GH31), displayed the typical 6 substrate specificity of GH31 α-glucosidases. The enzyme hydrolyzed not only α-(1→3)-but 7 also α-(1→2)-, α-(1→4)-and α-(1→6)-glucosidic linkages, and p-nitrophenyl α-glucoside. 8 SpGIIα displayed most catalytic properties of glucosidase II. Hydrolytic activity of the 9 terminal α-glucosidic residue of Glc2Man3-Dansyl was faster than that of This catalytic α-subunit also removed terminal glucose residues from native N-glycans 11 (Glc2Man9GlcNAc2 and Glc1Man9GlcNAc2) although the activity was low. 12