Abstract
Bacterial exopolysaccharide, dextran, primarily composed of α-(1→6)-linked d-glucosyl residues, is synthesized from α-(1→4)-glucan dextrin or sucrose through successive anomer-retaining transglucosylation reactions by dextran dextrinase (DDase) or dextransucrase, respectively. Although the structure-function relationship of dextransucrase has been extensively studied, that of DDase remains largely unknown. Herein, we revealed the Gluconobacter oxydans DDase structural basis through biochemical and structural analyses. The DDase comprises 1284 residues, with its N-terminal 902 residues being functionally essential. Crystal structure analysis of the minimal active DDase (Δ382C) complex with the pseudo-maltotetraose inhibitor, acarbose, revealed its homodimeric structure. A Δ382C protomer contains two β-sandwich domains, N1 and N2, and an (α/α)6-barrel domain A. Surprisingly, domains N2, A, and the helix-loop-helix connecting them structurally resemble those of bacterial anomer-inverting glucohydrolases in glycoside hydrolase family 15 (GH15). Domain N1 primarily forms intra- and inter-subunit domain interfaces. The DDase acarbose-binding residues in subsite -1 are conserved with GH15 glucohydrolases. The DDase Glu671 and Glu858 are positioned similarly to the GH15 glucohydrolase general acid and base catalysts, respectively. However, Glu858 is approximately 1.2 to 1.6 Å closer to the acarbose equivalent anomeric carbon, facilitating its role as a nucleophilic catalyst in the double displacement mechanism. The catalytic residue functions were biochemically confirmed using mutant enzymes. Spatial position of Glu858 is arranged by the local structure of the α11→α12 loop and subunit interactions involving domain N1. Enzymes classified in the same GH family catalyze reactions with different mechanisms, anomer-inverting or -retaining, due to differences in their catalytic residue spatial arrangement.