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

Pyruvyl modification of oligosaccharides is widely seen in both prokaryotes and eukaryotes. Although the biosynthetic mechanisms of pyruvylation have been investigated, enzymes that metabolize and degrade pyruvylated oligosaccharides are not well known. Here, we searched for a pyruvylated galactose (PvGal)-releasing enzyme by screening soil samples. We identified a Bacillus strain, as confirmed by the 16S ribosomal RNA gene analysis, that exhibited PvGal-ase activity toward p-nitrophenyl-β-D-pyruvylated galactopyranose (pNP-β-D-PvGal). Draft genome sequencing of this strain, named HMA207, identified three candidate genes encoding potential PvGal-ases, among which only the recombinant protein encoded by ORF1119 exhibited PvGal-ase activity. Although ORF1119 protein displayed broad substrate specificity for pNP sugars, pNP-β-D-PvGal was the most favorable substrate. The optimum pH for the ORF1119 PvGal-ase was determined as 7.5. A BLAST search suggested that ORF1119 homologs exist widely in bacteria. Among two homologs tested, BglC from Clostridium but not BglH from Bacillus showed PvGal-ase activity. Crystal structural analysis together with point mutation analysis revealed crucial amino acids for PvGal-ase activity. Moreover, ORF1119 protein catalyzed the hydrolysis of PvGal from galactomannan of Schizosaccharomyces pombe, suggesting that natural polysaccharides might be substrates of the PvGal-ase. This novel PvGal-catalyzing enzyme might be useful for glycoengineering projects to produce new oligosaccharide structures.

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This Article contains typographical errors in the Materials and Methods section under subheading ' Accession Numbers'. "The nucleotide sequences of the ORF1119, ORF4395 and ORF4971 genes have been deposited in the DDBJ/ EMBL/GenBank under the accession nos. LC306881, LC306883 and LC306885, respectively. " should read: "The nucleotide sequences of the ORF1119, ORF4395 and ORF4971 genes have been deposited in the DDBJ/ EMBL/GenBank under the accession nos. LC317050, LC317052 and LC317053, respectively. "