Levansucrase from Gluconacetobacter diazotrophicus. Key residues involved in levan synthesis

Levansucrase from Gluconacetobacter diazotrophicus (LsdA, EC 2.4.1.10) produces short-chain fructooligosaccharides, mainly 1-kestotriose, and levan polysaccharide from sucrose. By random mutagenesis, twenty-five LsdA residues were identified as being crucial for levan synthesis, with the substitution of these residues resulting in a decrease or complete elimination of levan synthesis. Fourteen residues (R171, H172, S228, R229, T243, F304, N306, E314, E327, R334, A369, D398, H419, and G432) were identified within the active site cavity, while eleven were dispersed across the protein. Saturation mutagenesis of H172 and R171 (-1 subsite) showed that the H172E/P mutant primarily exhibited sucrose hydrolysis, while the H172S and R171K-H172S variants were less affected in transfructosylation. Furthermore, HPAEC-PAD analysis revealed that the H172S and R171K-H172S variants synthesized 1-kestotriose, 6-kestotriose, 6G-kestotriose, and 1,1-kestotetraose. These variants had specific activity values similar to those of the native LsdA. However, the synthesis of 1,6-kestotetraose was found to be compromised, indicating a loss of the processive mechanism, suggesting that these variants have lost the ability to elongate via β-(2→6) links.