Insights into Broad-Specificity Starch Modification from the Crystal Structure of <i>Limosilactobacillus Reuteri</i> NCC 2613 4,6-α-Glucanotransferase GtfB

Abstract

GtfB-type α-glucanotransferase enzymes from glycoside hydrolase family 70 (GH70) convert starch substrates into α-glucans that are of interest as food ingredients with a low glycemic index. Characterization of several GtfBs showed that they differ in product- and substrate specificity, especially with regard to branching, but structural information is limited to a single GtfB, preferring mostly linear starches and featuring a tunneled binding groove. Here, we present the second crystal structure of a 4,6-α-glucanotransferase (<i>Limosilactobacillus reuteri</i> NCC 2613) and an improved homology model of a 4,3-α-glucanotransferase GtfB (<i>L. fermentum</i> NCC 2970) and show that they are able to convert both linear and branched starch substrates. Compared to the previously described GtfB structure, these two enzymes feature a much more open binding groove, reminiscent of and evolutionary closer to starch-converting GH13 α-amylases. Sequence analysis of 287 putative GtfBs suggests that only 20% of them are similarly "open" and thus suitable as broad-specificity starch-converting enzymes.