4,6-α-Glucanotransferase, a Novel Enzyme That Structurally and Functionally Provides an Evolutionary Link between Glycoside Hydrolase Enzyme Families 13 and 70

Abstract

ABSTRACTLactobacillus reuteri121 uses the glucosyltransferase A (GTFA) enzyme to convert sucrose into large amounts of the α-d-glucan reuteran, an exopolysaccharide. Upstream ofgtfAlies another putative glucansucrase gene, designatedgtfB. Previously, we have shown that the purified recombinant GTFB protein/enzyme is inactive with sucrose. Various homologs ofgtfBare present in otherLactobacillusstrains, including theL. reuteritype strain, DSM 20016, the genome sequence of which is available. Here we report that GTFB is a novel α-glucanotransferase enzyme with disproportionating (cleaving α1→4 and synthesizing α1→6 and α1→4 glycosidic linkages) and α1→6 polymerizing types of activity on maltotetraose and larger maltooligosaccharide substrates (in short, it is a 4,6-α-glucanotransferase). Characterization of the types of compounds synthesized from maltoheptaose by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), methylation analysis, and 1-dimensional1H nuclear magnetic resonance (NMR) spectroscopy revealed that only linear products were made and that with increasing degrees of polymerization (DP), more α1→6 glycosidic linkages were introduced into the final products, ranging from 18% in the incubation mixture to 33% in an enriched fraction. In view of its primary structure, GTFB clearly is a member of the glycoside hydrolase 70 (GH70) family, comprising enzymes with a permuted (β/α)8barrel that use sucrose to synthesize α-d-glucan polymers. The GTFB enzyme reaction and product specificities, however, are novel for the GH70 family, resembling those of the GH13 α-amylase type of enzymes in using maltooligosaccharides as substrates but differing in introducing a series of α1→6 glycosidic linkages into linear oligosaccharide products. We conclude that GTFB represents a novel evolutionary intermediate between the GH13 and GH70 enzyme families, and we speculate about its origin.