Substrate recognition of the catalytic α-subunit of glucosidase II from Schizosaccharomyces pombe

The recombinant catalytic alpha-subunit of N-glycan processing glucosidase II from Schizosaccharomyces pombe (SpGII alpha) was produced in Escherichia coli. The recombinant SpGII alpha exhibited quite low stability, with a reduction in activity to 3)-but also alpha-(1 -> 2)-, alpha-(1 -> 4)-, and alpha-(1 -> 6)-glucosidic linkages, and p-nitrophenyl alpha-glucoside. SpGII alpha displayed most catalytic properties of glucosidase II. Hydrolytic activity of the terminal alpha-glucosidic residue of Glc(2)Man(3)-Dansyl was faster than that of Glc(1)Man(3)-Dansyl. This catalytic alpha-subunit also removed terminal glucose residues from native N-glycans (Glc(2)Man(9)GlcNAc(2) and Glc(1)Man(9)GlcNAc(2)) although the activity was low

Molecular insight into regioselectivity of transfructosylation catalyzed by GH68 levansucrase and beta-fructofuranosidase

Glycoside hydrolase family 68 (GH68) enzymes catalyze beta-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, beta-fructofuranosidase (FFZm) and levansucrase (LSZm), encoded tandemly in the genome of Zymomonas mobilis, displayed different selectivity: FFZm catalyzed the beta-(2 -> 1)-trans-fructosylation (1-TF), whereas LSZm did both of 1-TF and beta-(2 -> 6)-trans-fructosylation (6-TF). We identified His79(FFZm) and Ala343(FFZm) and their corresponding Asn84(LSZm) and Ser345(LSZm) respectively as the structural factors for those regioselectivities. LSZm with the respective substitution of FFZm-type His and Ala for its Asn84(LSZm) and Ser345(LSZm) (N84H/S345A-LSZm) lost 6-TF and enhanced 1-TF. Conversely, the LSZm-type replacement of His79(FFZm) and Ala343(FFZm) in FFZm (H79N/A343S-FFZm) almost lost 1-TF and acquired 6-TF. H79N/A343S-FFZm exhibited the selectivity like LSZm but did not produce the beta-(2 -> 6)-fructoside-linked levan and/or long levanooligo-saccharides that LSZm did. We assumed Phe189(LSZm) to be a responsible residue for the elongation of levan chain in LSZm and mutated the corresponding Leu187(FFZm) in FFZm to Phe. An H79N/L187F/A343S-FFZm produced a higher quantity of long levanooligosaccharides than H79N/A343S-FFZm (or H79N-FFZm), although without levan formation, suggesting that LSZm has another structural factor for levan production. We also found that FFZm generated a sucrose analog, beta-D-fructofuranosyl alpha-D-mannopyranoside, by beta-fructosyltransfer to D-mannose and regarded His79(FFZm) and Ala343(FFZm) 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