A distinctive function of GH13_8 subfamily glycogen branching enzyme in Anaerococcus prevotii DSM 20548: Preference to create very short branches

Glycogen branching enzymes (GBEs; EC 2.4.1.18) are essential for forming α-1,6-O-glycosidic branches in starch modification and glycogen biosynthesis. They are classified into glycoside hydrolase (GH) families 13 and 57. GH13 GBEs are further divided into subfamilies GH13_9, containing predominantly sequences from bacteria, and GH13_8, comprising sequences from both bacteria and eukaryotes. So far, only three eukaryotic GH13_8 enzymes have been studied in detail while no crystal structures or functional activities of prokaryotic GH13_8 GBEs have been reported. In this study, the GH13_8 and GH13_9 GBE of Anaerococcus prevotii (Ap) were studied in detail. It was shown for the first time that this prokaryotic GH13_8 GBE is active on amylose and creates α-1,6-O-glycosidic linked branches. In contrast to GH13_9 GBEs, the ApGBE13_8 is active on very short oligosaccharides ranging from DP2 to DP5 (degree of polymerization) transferring glucose or maltose. The preference for short oligosaccharides might be correlated with the presence of two short beta stranded loops at position 131 and 509. These loops may function like a 'door,' dynamically adjusting to the donor chain, affecting branch length and cleavage specificity. These findings reveal ApGBE13_8's distinct function, advance GH13_8 research, and suggest potential applications for GH13_8 GBEs in starch modification.