4,3-α-Glucanotransferase, a novel reaction specificity in glycoside hydrolase family 70 and clan GH-H

Lactic acid bacteria possess a diversity of glucansucrase (GS) enzymes that belong to glycoside hydrolase family 70 (GH70) and convert sucrose into alpha-glucan polysaccharides with (alpha 1 -> 2)-, (alpha 1 -> 3)-, (alpha 1 -> 4)-and/or (alpha 1 -> 6)-glycosidic bonds. In recent years 3 novel subfamilies of GH70 enzymes, inactive on sucrose but using maltodextrins/starch as substrates, have been established (e.g. GtfB of Lactobacillus reuteri 121). Compared to the broad linkage specificity found in GSs, all GH70 starch-acting enzymes characterized so far possess 4,6-alpha-glucanotransferase activity, cleaving (alpha 1 -> 4)-linkages and synthesizing new (alpha 1 -> 6)-linkages. In this work a gene encoding a putative GH70 family enzyme was identified in the genome of Lactobacillus fermentum NCC 2970, displaying high sequence identity with L. reuteri 121 GtfB 4,6-alpha-glucanotransferase, but also with unique variations in some substrate-binding residues of GSs. Characterization of this L. fermentum GtfB and its products revealed that it acts as a 4,3-alpha-glucanotransferase, converting amylose into a new type of alpha-glucan with alternating (alpha 1 -> 3)/(alpha 1 -> 4)-linkages and with (alpha 1 -> 3,4) branching points. The discovery of this novel reaction specificity in GH70 family and clan GH-H expands the range of alpha-glucans that can be synthesized and allows the identification of key positions governing the linkage specificity within the active site of the GtfB-like GH70 subfamily of enzymes.