Polysaccharide Synthesis of the Levansucrase SacB from Bacillus megaterium Is Controlled by Distinct Surface Motifs

Abstract

Despite the widespread biological function of carbohydrates, the polysaccharide synthesis mechanisms of glycosyltransferases remain largely unexplored. Bacterial levansucrases (glycoside hydrolase family 68) synthesize high molecular weight, ␤-(2,6)-linked levan from sucrose by transfer of fructosyl units. The kinetic and biochemical characterization of Bacillus megaterium levansucrase SacB variants Y247A, Y247W, N252A, D257A, and K373A reveal novel surface motifs remote from the sucrose binding site with distinct influence on the polysaccharide product spectrum. The wild type activity (k cat ) and substrate affinity (K m ) are maintained. The structures of the SacB variants reveal clearly distinguishable subsites for polysaccharide synthesis as well as an intact active site architecture. These results lead to a new understanding of polysaccharide synthesis mechanisms. The identified surface motifs are discussed in the context of related glycosyltransferases.

Crystal Structure of Inulosucrase from Lactobacillus: Insights into the Substrate Specificity and Product Specificity of GH68 Fructansucrases

“…and + 2 but also provided particularly by higher subsites (+ 3, + 4, …, etc.). First, mutations targeting levansucrase residues in subsites + 1 and + 2 (e.g., R360 in BsSacB, 17 R370 and N252 in BmSacB, 9,18 and R331 in B. subtilis QB252 levansucrase 19 ) altered the levan/oligosaccharide product ratio and the transfructosylation/hydrolysis ratio, but not the linkage type of the products. Second, the structure of InuJ with the FOS-type 1-kestose bound in subsites − 1 to +2 shows that all observed interactions between enzyme and trisaccharide are provided by conserved residues.…”

“…15,16 Product specificity has been addressed in many studies on bacterial levansucrases, 9,15,[17][18][19][20] but so far, it has been poorly understood. Mutation of residues lining acceptor subsites has been shown to affect the transfructosylation/hydrolysis ratio and the FOS/levan polymer ratio, and thus the length of the synthesized products (see van Hijum et al 1 and Lammens et al 6 for reviews).…”

“…3 GH68 is part of Clan-J, together with family GH32, which includes yeast, plant, and fungal fructosyltransferases. [4][5][6] To date, three-dimensional (3D) structures of bacterial GH68 levansucrases from Bacillus subtilis 7,8 (BsSacB), Bacillus megaterium (BmSacB), 9 and Gluconacetobacter diazotrophicus 10 (LsdA) have been determined. For GH32, 3D structural information is available for the fructosyltransferase from Aspergillus japonicus (AjFT), 11 which synthesizes inulin-type fructo-oligosaccharides (FOSs), as well as for several GH32 fructan hydrolases.…”

Recent novel applications of levansucrases

“…The enzyme produces both levan and FOSs (Homann et al 2007). The crystalstructure has been determined, and it is indicated that levan synthesis is controlled by distinct surface motifs (Strube et al 2011 Crude enzyme The optimum pH and temperature for levan production are pH 6.0 and 37°C, respectively. The molecular mass of the produced levan is between 4 and 5×10 3 Da, and the highest production is 100 g L −1 after 16 h of reaction under optimum conditions.…”

Structural and Functional Basis for Substrate Specificity and Catalysis of Levan Fructotransferase

“…In addition to their role as an energy reserve, plant fructans also function in cold and drought tolerance (1)(2)(3). Their biosynthetic pathways in bacteria are relatively well established; in these pathways, a single multifunctional enzyme of levansucrase (4,5) and inulosucrase (6) is responsible for the synthesis of levan and inulin, respectively, with sucrose as a substrate. However, those pathways have not yet been conclusive in plants (7).…”