Functional and structural characterization of alpha-(1-2) branching sucrase derived from DSR-E glucansucrase

摘要: ΔN123-glucan-binding domain-catalytic domain 2 (ΔN123-GBD-CD2) is a truncated form of the bifunctional glucansucrase DSR-E from Leuconostoc mesenteroides NRRL B-1299. It was constructed by rational truncation GBD-CD2, which harbors second catalytic DSR-E. Like this variant displays α-(1→2) branching activity when incubated with sucrose as glucosyl donor and (oligo-)dextran acceptor, transferring residues to acceptor via ping-pong bi-bi mechanism. This allows formation prebiotic molecules containing controlled amounts linkages. The crystal structure apo sucrase ΔN123-GBD-CD2 solved at 1.90 Å resolution. protein adopts unusual U-shape fold organized in five distinct domains, also found GTF180-ΔN GTF-SI glucansucrases glycoside hydrolase family 70. Residues forming subsite −1, involved binding residue catalysis, are strictly conserved both ΔN123-GBD-CD2. Subsite +1 analysis revealed three (Ala-2249, Gly-2250, Phe-2214) that specific Mutation these corresponding showed Ala-2249 Gly-2250 not directly substrate regiospecificity. In contrast, mutant F2214N had lost its ability branch dextran, although it still active on alone. Furthermore, loops belonging domains A B upper part gorge These distinguishing features proposed be correct positioning dextran allowing branches.