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

Abstract

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 (FFZm) and levansucrase (LSZm), encoded tandemly in the genome of Zymomonas mobilis, displayed different selectivity: FFZm catalyzed the β-(2→1)-transfructosylation (1-TF), whereas LSZm did both of 1-TF and β-(2→6)-transfructosylation (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 β-(2→6)-fructoside-linked levan and/or long levanooligosaccharides 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, β-D-fructofuranosyl α-D-mannopyranoside, by β-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.

Keywords: glycoside hydrolase family 68; levansucrase; regioselectivity; transfructosylation; β-d-fructofuranosyl α-d-mannopyranoside; β-fructofuranosidase.